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The role of brca1 domains and motifs in tumor suppression
h [electronic resource] /
by Aneliya Velkova.
[Tampa, Fla] :
b University of South Florida,
Title from PDF of title page.
Document formatted into pages; contains 154 pages.
(Ph.D.)--University of South Florida, 2011.
Includes bibliographical references.
Text (Electronic dissertation) in PDF format.
ABSTRACT: ABSTRACT Individuals that carry deleterious mutations in the breast and ovarian cancer susceptibility gene 1 (BRCA1) have much more elevated risk to develop breast and/or ovarian cancer than the individuals from the general population. The BRCA1 gene product has been implicated in several aspects of the DNA damage response, but its biochemical function in these processes has remained elusive. In order to probe BRCA1 functions we conducted a yeast two-hybrid screening to identify interacting partners to a conserved motif (Motif 6) in the central region of BRCA1. In this dissertation, we report the identification of the actin-binding protein Filamin A (FLNA) as a BRCA1 partner and demonstrate that FLNA is required for the efficient regulation of DNA repair process at its early stages. Cells lacking FLNA display a diminished ionizing radiation (IR)-induced BRCA1 focus formation and a slow kinetics of Rad51 focus formation. In addition, our data demonstrate that FLNA is required to stabilize the interaction between DNA-PK holoenzyme components such as DNA-PKcs and Ku86 in a BRCA1-independent manner. Our data are consistent with a model in which the absence of FLNA compromises homologous recombination and non-homologous end joining. Our findings have implications for our understanding of the response to irradiation-induced DNA damage.
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Monteiro, Alvaro .
Dna Damage Response
Varints Of Uncertain Signifficance
x Molecular Biology
t USF Electronic Theses and Dissertations.
The Role of BRCA1 Domains and Motifs in Tumor Suppression by Aneliya Velkova A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Cellular and Molecular Microbiology College of Arts and Sciences University of South Florida Major Professor: Alvaro N.A. Monteiro, Ph.D. Jiandong Chen, Ph.D. Gary W. Reuther, Ph.D. William Dalton, MD, Ph.D. Date of Approval: May 16, 2011 Keywords: Breast Cancer, Variants of Uncertain Signi ficance, DNA Damage response, Filamin A, DNAPKcs Copyright 2011, Aneliya Velkova
DEDICATION To the memory of my grandfathers, Petar Rangelov and Jordan Velkov, and to my family.
ACKNOWLEDGEMENTS First and foremost, I would li ke to thank my mentor Dr. Alvaro Monteiro. In my experience, the most important thing in graduate school is finding the right mentor. Everything, that I know now starting from how to work with yeast to how to write grants and to be a critical thinker, I ow e to Dr. Alvaro Monteiro. I am grateful for the inspiring conversations we had about developmental biology, science history, BRCA1 and breast cancer, and life in general. Overall, we should not forget that Biology is the science for studying LIFE. I will a lways say Thank you! to Dr. Monteiro for giving me a head start and for having the rare gift of being a teacher. I would also like to thank my committee members Dr. Jiandong Chen, Dr. Gary Reuther, Dr. William Dalton, the Cancer Biology Ph.D. Program Dir ector Dr. Kenneth Wright, and Dr. Mark Alexandrow for their constant guidance and support over the years. In addition, I am grateful to Dr. William Foulkes for taking the time to serve as an outside chair at my defense. I would like to acknowledge former l ab members Alyson Freeman, Virna Dapic, Marcelo Carvalho, Jonathan Rios Doria, and Sylvia Marsillac and current members Melissa Price, Nick Woods, Huey Nguyen, Anxhela Gjushi, and Xueli Li for their help, constant support, great scientific discussions (som etimes at 10pm at night), and for making the lab feel like home. Moreover, I thank Cathy Gaffney for her support, understanding, and enormous work that she does for the Cancer Biology Ph.D. program. I am also grateful to Scott Mears for his enormous profes sionalism and understanding during the grant submission
process. I would never survive the bureaucracy without Cathy's and Scott's help. I want to extent a special thank you to Dimitar who understands how important science is for me, does not hesitate to c ome with me to collect time points, even at 2am on Saturday, and is constant inspiration and support.
i TABLE OF CONTENTS LIST OF FIGURES iv LIST OF TABLES vii LIST OF ABBREVIATIONS viii ABSTRACT x INTRODUCTION 1 BRCA1 and Cancer Predisposition 1 Hereditary Predisposition to Breast and Ovarian Cancer 1 Early Findings in Hereditary Breast and Ovarian Cancer 3 Breast and Ovarian Cancer Risk Associated with BRCA1 5 Contribution of BRCA1 to Breast and Ovarian Cancer 7 BRCA1 Is a Tumor Suppressor Gene 8 Spectrum of Mutations in the BRCA1 Gene 10 Genetic Testing for BRCA1 and the Variants of Uncertain Significance (VUSs) Problem 11 How to determine the Associati on of a Genetic Variant with a Disease? 12 Why Are Studies on BRCA1 Important? 18 The DNA Damage Response (DDR) Network 19 DDR: DNA Repair and Cell Cycle Checkpoints 19 Sensors, Signal Transducing Kinases, Effector Kinases, and Effector Proteins 23 Checkpoint Mediators 25 DDR: Spatiotemporal Regulation in Specialized Chromatin Domains 28 BRCA1: Structure and Functions 30 The Structure of BRCA1 30 BRCA1: Biochemical Activities and Biological Processes 35 G1 Checkpoint 36 Intra-S-Phase Checkpoint 37
ii G2/M Checkpoint 41 Spindle Assembly Checkpoint 45 DNA Repair by HR 46 DNA Repair by NHEJ 50 Hypotheses for the Tissue Specific BRCA1 Functions 52 Summary and Rationale 53 MATERIALS AND METHODS 55 Constructs and Cloning 55 Yeast Two Hybrid Assays 56 Cell Lines and Transfections 57 Antibodies 57 Immunoprecipitation, Pull-Downs, Western Blot Analysis, and Densitometry 58 Immunofluorescence 60 Comet Assay 61 RESULTS 62 Characterization of the Interaction between FLNA and BRCA1 62 Identification of FLNA as a Binding Partner of BRCA1 Motif 6 in Yeast 62 Interaction between FLNA and BRCA1 in Mammalian Cells 63 BRCA1 Binding to FLNA is Mediated Primarily by BRCA1 Motif2 not Motif 6 68 BRCA1 Variant Y179C, Found in Breast and Ovarian Cancer Patients, Disrupts the Binding between BRCA1 and FLNA 77 Analysis of the DNA Damage Response Signaling in FLNA Positive and Negative Cell Lines 79 FLNA Deficiency Does not C ause a Defect in Sensing DNA Damage 79 The Defect in FLNA-Deficient Cells Is Not Restricted to Ionizing Radiation 82 FLNA Deficiency Impairs BRCA1 and Rad51 Foci Formation 91 The Lack of FLNA Leads to Accumulation of ssDNA after DNA Damage 95
iii Analysis of the Expression of BRCA1-Interacting Fragment of FLNA and FLNA-Interacti ng Fragment of BRCA1 in FLNA Positive Cell Line 98 Expression of BRCA1-Interacting Fragment of FLNA Phenocopies Loss of FLNA 98 Expression of BRCA1-Interacting Fragment of FLNA Phenocopies Loss of FLNA 102 Analysis of the Effect of FLNA on DNA-PKcs and Ku86 Interaction 104 FLNA is Required for Efficient Interactions Between DNA-PKcs and Ku86 104 Lack of FLNA Leads to an Increase in End Joining Activity 110 DISCUSSION 112 LIST OF REFERENCES 119 ABOUT THE AUTHOR END PAGE
iv LIST OF FIGURES Figure 1. Breast cancer suscep tibility genes. 4 Figure 2. Figure 2. Breast cancer susceptibility gene products and the DDR pathway. 21 Figure 3. DNA repair by homologous recombination and non homologous end joining. 22 Figure 4. Current model for the BRCA1 recruitment to the IRIF. 26 Figure 5. Structure of BRCA1. 32 Figure 6. Cell cycle stages and participation of BRCA1 in cell cycle checkpoints. 38 Figure 7. The intra-S-phase checkpoint signaling. 39 Figure 8. The G2/M checkpoint signaling. 42 Figure 9. BRCA1 Motif 6 binds to Filamin A (aa 2477-2674) in yeast. 64 Figure 10. BRCA1 and Filamin A interact in mammalian cells. 65 Figure 11. FLNA interacts with BRCA1 in the nucleus. 66 Figure 12. FLNA directly binds to BRCA1 in vitro. 67 Figure 13. Growth curve in me dium lacking leucine and tryptophane. 69 Figure 14. Interaction between pGBKT7 Motif 6 mutants (aa 845-869) and pGAD424 FLNA (aa 2477-2647) constructs. 70 Figure 15. Expression of BRCA1 fragments in 293FT cell line. 71
v Figure 16. FLNA interacts with multiple sites in BRCA1. 72 Figure 17. GST-pull down experi ments show that GST-BRCA1 fragments 1, 3, and 4 can precipitate endogenous FLNA (WB FLNA). 73 Figure 18. FLNA binds to aa 141-240 in BRCA1. 74 Figure 19. FLNA binds to aa 160-190 in BRCA1. 75 Figure 20. Introduction of BRCA 1 Y179C mutation significantly reduces BRCA1 interaction to FLAG-FLNA aa 2477-2647 and to endogenous FLNA. 78 Figure 21. FLNA-null cells are deficient in repair. 80 Figure 22. Neutral comet confir ms that FLNA-null cells are deficient in repair. 81 Figure 23. FLNA-null cells show no impairment in activating the DNA damage response. 84 Figure 24. CHK2, CHK1, and NBS1 activation as measured by pT68-CHK2, pS317-CHK1, and pS343-NBS1, respectively is not compromised in FLNA cells. 85 Figure 25. Effects of the CPT treatment on the DNA damaging signaling in FLNA-deficient and proficient cell lineATM, CHK2 and DNA-PKcs activation. 86 Figure 26. Effects of CPT treatment on the DNA damage response signaling in FLNA-deficient and proficient cell lineCHK1 and NBS1 activation. 87 Figure 27. Effects of CPT treatment on the DNA damage response signaling in FLNA-deficient a nd proficient cell line-RPA phosphorylation. 88 Figure 28. Effects of HU treatment on the DNA damage response signaling in FLNA-deficient and proficient cell lineCHK1, CHK2 and NBS1 activation. 89 Figure 29. Effects of HU treatme nt on the DNA damage response signaling in FLNA-deficient a nd proficient cell line-RPA phosphorylation. 90
vi Figure 30. FLNA-null cells show no impairment in recruiting DNA damage response factors to IR-induced foci -H2AX and NBS1. 92 Figure 31. Recruitment of DNA damage response mediator proteins 53BP1 (red, top panel) and MDC1 (green, middle panel) and BRCA1 to foci. 93 Figure 32. Recruitment of RPA and Rad51 to IR induced nuclear foci. 94 Figure 33. FLNA-deficient cells present with large chromatin -bound RPA foci at 24h after IR. 96 Figure 34. Criteria for scoring foci positive cells. 97 Figure 35. Expression of BRCA 1-interacting fragment of FLNA in FLNA is unable to reverse the checkpoint recovery defect. 99 Figure 36. Expression of BRCA1interacting fragment of FLNA phenocopies loss of FLNA. 100 Figure 37. HCT166 cells stably expressing FLAG FLNA-Bf display a phenotype similar to FLNA cells. 101 Figure 38. Expression of FLNA-i nteraction fragment of BRCA1 Y179C mutant does not phenocopy loss of FLNA. 103 Figure 39. FLNA interacts in vivo with DNA-PKcs. 106 Figure 40. FLNA mediates DNA-PKcs interaction to Ku86. 107 Figure 41. Loading of Ku86 onto chromatin after DNA damage is increased in FLNA cells. 108 Figure 42. BRCA1 is not required for the stabilization of the interaction between DNA-PKcs and Ku86. 109 Figure 43. Lack of Filamin A lead s to an increase in end joining Activity. 111
vii LIST OF TABLES Table 1. Lifetime risk for br east and ovarian cancer among carriers of BRCA1 mutations. 6 Table 2. Mouse models for BRCA1 deficiency. 9 Table 3. BRCA1 domains and motifs. 31 Table 4. Comparisons of different assays for BRCA1 repair function. 51
viii LIST OF ABREVIATIONS ATM Ataxia Talangiectasia Mutated ATR Ataxia Talangiectasia Mutated and Rad3 Related ATRIP ATR Iteracting Protein BACH1 BRCA1 associated C-terminal Helicase 1 BARD1 BRCA1 Associated RING Domain One Gene BRCA1 Breast and Ovarian Can cer Susceptibility Gene One BRCA2 Breast and Ovarian Can cer Susceptibility Gene Two BRCT BRCA1 Carboxy Terminal Domain CDK Cyclin Dependent Kinase CHK1 Checkpoint Kinase One Gene CHK2 Checkpoint Kinase Two Gene CPT Campthothecin DDR DNA Damage Response DNA-PKcs DNA Dependent Protei n Kinase Catalytic Subunit DSB Double Strand Breaks FHA Forkhead Associated Domain FLNA Filamin A GADD45a Growth Arrest and DNA Damage Inducible 45 alpha
ix HR Homologous Recombination HU Hydroxyurea IP Immunoprecipitation IR Ionizing Radiation LOH Loss of Heterozygosity MRE11 Meiotic Recombination 11 Homologue MRN MRE11, RAD50, NBS1 NBS1 Nijmegen Breakage Syndrome One NHEJ Non-Homologous End Joining NLS Nuclear Localization Sequence Rap80 Receptor Associated Protein 80 RDS Radioresistant DNA Synthesis RING Really Interesting New Gene Domain RPA Replication Protein A ssDNA Single Stranded DNA SMC1 Structural Maintenance of Chromosomal Protein 1 UV Ultraviolet VUS Variant of Uncertain Significance WB Western Blot WHO World Health Organization 9-1-1 complex Rad9-Rad1-Hus1
x ABSTRACT Individuals that carry deleterious muta tions in the breast and ovarian cancer susceptibility gene 1 ( BRCA1 ) have much more elevated ri sk to develop breast and/or ovarian cancer than the individuals from the general population. The BRCA1 gene product has been implicated in several as pects of the DNA damage response, but its biochemical function in these processes has re mained elusive. In order to probe BRCA1 functions we conducted a yeast tw o-hybrid screening to identify interacting partners to a conserved motif (Motif 6) in the central re gion of BRCA1. In this dissertation, we report the identification of the actin-binding protein Filamin A (FLNA) as a BRCA1 partner and demonstrate that FLNA is required for the effi cient regulation of DNA repair process at its early stages. Cells lacki ng FLNA display a diminished i onizing radiation (IR)-induced BRCA1 focus formation and a slow kinetics of Rad51 focus formation. In addition, our data demonstrate that FLNA is required to stabilize the interaction between DNA-PK holoenzyme components such as DNA-PKcs and Ku86 in a BRCA1-independent manner. Our data are consistent with a model in which the absence of FLNA compromises homologous recombination and non-homologous end joining. Our findings have implications for our understanding of the response to irradiation-induced DNA damage.
1 INTRODUCTION BRCA1 and Cancer Predisposition Hereditary Predisposition to Breast and Ovarian C ancer A ccording to the American Cancer Society, 207,090 women and 1,970 men were diagnosed with breast cancer in 2010 i n the US alone. Moreover, b reast canc er is among the most frequent malignancies affecting women worldw ide. Germline mutations in the breast and o varian cancer predisposition gene 1 (BRCA1) are responsible for the majority of early onset breast and ovarian cancers arising in families with mult iple cases. Furthermore, it has been shown that mutations in BRCA1 and BRCA2 genes occur with fr e quency of 1:250, which translates into 250 000 women in the US who are carriers  Therefore, it is crucial to identify individuals at risk early. These individuals and their families have the options of increased surveillance, chemoprevention or prophylact ic surgeries all of which can potentially reduce the possibility of developing cancer. The cloning of BRCA1 made early identification of individuals at risk possible by genetic testing. However, there are many difficulties that leave around 10% of the wo men who undergo testing with uninformative results due to the finding of BRCA1 variants for
2 which the cancer association is not known. These variants are called variants of uncertain significance (VUSs). Moreover in minority populations the percentage of women, r e ceiving uninformative results is significantly higher (35 50%) making it a health dispari ty issue that needs to be addressed These women do not have all the information to make informed clinical decisions. In addition, BRCA1 is inactivated in som e sporadic breast cancer cases suggesting an important role for BRCA1 in breast cancer in general. As far as its presentation in families, breast and ovarian cancer can be considered as hereditary, familial, or sporadic. Ascertainment criteria for heredit ary (inherited) breast and/or ovarian cancer syndrome often include but are not limited to: early age of cancer onset, multiple affected individuals within a family, development of bilateral di s ease, and male individuals with breast cancer. Hereditary brea st and ovarian cancer cases have strong family history for the disease and can often be attributed to mutations in genes such as BRCA 1 and BRCA2 In familial cancer there is a patter n in the family, pointing a hereditary component However, these cases ha ve not been ascribed to mut a tions in any of the known genes. Sporadic cancer can be defined as a type of cancer which arises in individuals that usually do not have family history and the disease has not been attributed to any known genetic factor. Howev er, we should point out that this classification is arbitrary and is often difficult to make a clear cut distinction between di f ferent classes. In this thesis we focus on BRCA1 tumor syndrome with autosomal do minant trait and markedly increased susceptibility to breast and ovarian tumors, due to germ line mutations in the BRCA1 gene. Additional
3 o r  It is estimated that 5 10% of all breast cancer ca ses are hereditary (Figure 1). Approximat e ly 30 45% of the hereditary cases can be at tributed to BRCA1 and BRCA2 mutations  According to our current understanding here ditary breast cancer can be assigned to three groups of susceptibility alleles: genes that have high ( e.g. BRCA1 and BRCA2 ), moderate ( e.g. ATM, BRIP1, CKEK2, and PALB2 ), and low penetrance alleles ( e.g. FGFR2 TNRC9 and MAP3K1 )  Early Findings in Hereditary Breast and Ovarian Cancer The first report about hereditary breast cancer dates back to 1866. It was made by the French surgeon Paul Broca  who repo rted that in five generations of fam i ly 10 out of the 24 women died from breast cancer  Another important discove ry in breast cancer research happened in 1950 when Gardner and Stephens examined 668 i n dividuals from a Utah family. Their findings showed that the frequency of breast cancer was 20 times higher in that family than in the general population of Utah over the age of 30  The researchers  The nature of ca n cer. These studies explored the role of environment and family history in breast
4 Figure 1. Breast cancer su sceptibility genes. Hereditary breast cancer accounts for 5 10% of all breast cancer cases. BRCA1 and BRCA2 are responsible for appro x imately 30 45% of hereditary breast cancer cases. Mutations in ATM CHK2 PTEN PALB2, RAD50, NBS1, BRIP1, and TP53 explai n some of the remaining cases. The rest of the hereditary cases have not been attributed to the any known susceptibility genes. (Adapted from  ).
5 cancer predisposition. Therefore, Lynch and colleagues decided to study 34 families, purposef ully selected because they had two or more members affected with breast cancer. Their findings showed that in some of those families the significant predisposition was not only for breast cancer, but also for other malignancies  They concluded that mu l tiple factors including interactions of different genetic and environmental factors could contribute to cancer predisposition  Further, Newman et. al analyzed data from 1579 families with multiple breast cancer cases. Based on this analysis, the researchers offered a mo del for the explanation of the breast cancer frequent occurrence  According to that model, inherited breast cancer predisposition was caused by an autosomal dominant allele with high but incomplete penetrance  That study was followed by mapping the gene responsible for early onset breast c ancer to chromosome 17q21 [11, 12] Finally, the seminal discovery on hereditary breast and ovarian cancer happened in 1994 when the first breast and ovarian cancer susceptibility gene BRCA1 was cloned  Breast and Ovarian Cancer Risk A ssociated with BRCA1 Germline mutations in BRCA1 are responsible for 30 45% of all hereditary breast cancer cases and around 80% of the cases that have multiple breast and ovarian cancers  Therefore, BRCA1 is the major locus responsible for the breast and ovarian cancer predisposition. The lifetime risks for developing breast and ovarian cancer in the general population are estimated to be 12.7 % and 1.3% respectively ( American Cancer Society
6 2010 ). Meanwhile, women who carry deleterious mutations in BRCA1 have the lifetime risk of developing breast cancer of 36 82% and 16 49% for ovarian cancer (Table 1). The wide range of those risk estimates c an be explained by differences in the study design, populations, and the age limit used to calculate lifetime risk (Table 1). Nevertheless, the risk for women who are carriers of deleterious mutations in BRCA1 to develop breast and/or ovarian cancer is fiv e to ten times higher than the risk for the women in the gene r al population. Finally, deleterious mutations in BRCA1 are accepted to be the best known predictor for breast cancer risk [15, 16] Table 1. Lifetime r isk for breast and ovarian cancer among carriers of BRCA1 m u tations Breast cancer risk % Ovarian cancer risk % Additional information Refs 65 39 Combines 22 studies  69 49 AJ (Ashkenazi Jewish)  43 67 14 33 AJ  36 AJ  82 Selection for strong family history  37 AJ  68 36 Patients diagnosed with ovarian cancer in Ontario, Canada 1995 96  46 AJ  56 16 AJ  59.9 AJ, unselected for family history  72.8 40.7 Risk, estimated up to the age of 70 
7 In contrary, in sporadic breast and ovarian cancer cases the situation is different: somatic BRCA1 mutations are rare [27, 28] but epigenetic inactivation by promoter hypermethylation [29 31] or decreased expression of BRCA1 [32, 33] have been found in sporadic tumors. This suggests that BRCA1 may also play a role in sporadic brea st and ovarian cancer development Contribution of BRCA1 to Breast and Ovarian C ancer Breast cancer is a heterogeneous disease. Based on gene expression patterns, it can be classified into five distinct groups: luminal A and B, normal breast like, ERBB2 and basal like breast carcinomas  The majority of BRCA1 related breast tumors can be classified as basal like type breast carcinomas [34, 35] The a nalysis of gene exp re s sion profiles of BRCA1 related breast tumors led to the conclusion that they are more likely to be estrogen receptor alpha negative, progesterone receptor negative, and Her2/neu negative than the sporadic ones [34 37] (also referred as triple negative breast cancer). Moreover, mutations in TP53 are more frequent in BRCA1 related breast tumors than in the sporadic ones [38, 39] Strikingly, triple negative breast cancer occu rs more frequently in pre menopausal African American women when compared to the age matched female white Americans  Finally, triple negative breast cancer in African Americans phenotypically share the same characteristics as BRCA1 related breast cancer 
8 Similar to the studies on the breast cancer, attempts have been made to class ify ovarian tumors u sing a gene expression profile  Unfortunately, the sample size of these studies was small. Therefore, they did not have sufficient statistical power, which precluded the classification o f ovarian cancer. Based on histology, over 90% of BRCA1 linked ovarian tumors are serous adenocarcinomas  BRCA1 I s a Tumor Supp ressor G ene Inheritance of one defective BRCA1 copy is enough to cause cancer predispos i tion, but in order for cancer to develop, the remaining wild type allele needs to be inact i vated  Therefore, BRCA1 hit hypothe for the tumor suppressor gene: both alleles are inactivated in cancer  In addition, mouse models support the fact that BRCA1 is a tumor suppresso r. Although inactivation of both Brca1 alleles causes embryonic lethality [45 48] specific inactivation of Brca1 in mammary gland cells leads to the tumor development [49 52] Interestingly, unlike heter o zygous women, heterozygous mice did not develop tumors  However, mamm ary t u mors resembling the basal like breast tumors of BRCA1 mutation carriers were detected after conditional inactivation of Brca1 in the mammary glands [50, 51, 53] A summary of Brca1 related mouse models and their phenotypes is shown on Table 2. In contrast to conditional mouse models developed to study the role of BR CA1 in breast cancer, there are very few mouse models on the ovarian cancer progression [54 56] In the first mouse model for Brca1 linked ovarian cancer, Brca1 was conditionally
9 inactivated in the ovarian granulosa cells  The mice developed cystic tumors, which are thought to precede ovarian cancer  Such tumors carried normal Brca1 alleles, which indicated a paracrine mechani sm in ovarian cancer development Brca1 influences tumor development in the ovarian surface epithelium indirectly through granulosa cells  Table 2. Mouse models for BRCA1 deficiency (adapted from  ) Allele Phenotype Cre transgene Refs Brca1 ex2 death at e7.5  Brca1 5 6 death at e7.5  Brca1 11 death at e7.5 9.5  Brca1 223 763 death at e10 13.5  Brca1 ko death at e7.5 8.5  Brca1 S971A none  Brca1 Tr lymphomas, sarcomas, breast cancer  Brca1 1700T death at e10.5  Brca1 F5 6 thymocyte specific Lck Cre  Brca1 Co/ death at e12.5  Brca1 11 lymphoma, ovarian tumors  Brca1 F11/F11;p53+/ mammary tumors MMTV Cre or WAP Cre  Brca1 F11/F11;p53F5 6/p53F5 6 mammary tumors WAP Cre c  Brca1 F22 24/F22 24;p53+/ basal like breast cancer BLG Cre  Brca1 F5 13/F5 13;p53F2 10/F2 10 basal lik e breast cancer K14 Cre  Brca1 F2/F2 basal like breast cancer WAP Cre 
10 In the other mouse model, Brca1 was inactivated in the ovarian surface epithelial cells, whic h resulted in pre neoplastic changes, but not ovarian cancer  In concl u sion, it is not known how well these models represent the ovarian cancer in humans, and the role of BRCA1 in the transformation of ovarian surface epithelial cells is unclear. Nevertheless, the Brca1 related mouse models support the evidence that BRCA1 is a t u mor suppressor gene and set the stage for studying the mechanisms by which BRCA1 suppresses tumor formation at the organism level. Spe ctrum of Mutations in the BRCA1 G ene The mutation spectrum of BRCA1 varies in different populations. For example, genetically heterogeneous populations such as those in the United States, the United K ingdom, and Canada display big differences in documente d BRCA1 mutations ( reviewed in   ) On the other hand in geographically or historically isolated popul a tions like Icelanders or Ashkenazi Jews the majority of hereditary breast and o varian cancer can be attributed to founder mutations  Founder mutations are specific mutations that appear repeatedly in ethni cally defined groups because of genetic drift  For exam ple, three founder mutations in BRCA1 (185 delAG HGVS nomenclature NC_000017.9: g. 38529572_ g.38529571delAG and 5382 ins C NC_000017.9: g.38462606dupC ) and BRCA2 (6174 del T NC_000013.9:c.24822delT ) genes are responsible for most of breast and ovarian canc er cases in individuals from Ashkenazi Jewish descent  Founder mutations in BRCA1 have also been found in populations
11 from Germany, Norway, Poland and Sweden  The identification of founder mutations facilitates genetic testing for hereditary breast and ovarian cancer in the studied population s by decreasing the cost and the time of the test. Genetic T esting for BRCA1 and the Variant o f Uncertain Significance (VUS) P roblem The cloning of BRCA1 and BRCA2 made the early identification of individuals at risk possible by standard genetic testing    which is done by direct sequen c ing  The results of genetic testing inf luence clinical decisions such as increased su r veillance, prophylactic surgery, chemoprevention, and response to therapy (both radiati on and chemotherapy). In the US genetic testing for BRCA1 and BRCA2 is done by Myriad Genetics Laboratories and has three outcomes: (1) Negative ("No mutation detected" or "Favor polymorphism"). In the case of BRCA1 this means that no cancer associated (deleterious) mutation was detected This result is informative for the proband only if a BRCA1 mutation is found in other affected family members. In this case, it excludes the person that is undergoing testing. However, it is important to point out that a negative result can be due to changes that cannot be d e tected by direct sequencing such as large rearrangements that lea d to deletion of whole exons and mutations in the regulatory regions  For these cases there is an additional test that can be applied called BART (BRCAanalysis Rearrangement Test), which tests for a panel of rearrangements in BRCA1 and BRCA2 genes. Another possible reason for an indi vidual with a family histo ry of breast and ovarian cancer to get a negative result is
12 that the family might have a mutation in other breast cancer predisposing genes such as CHEK2 and ATM  A recent study has revealed that even when first deg ree relatives of women, carriers of BRCA1 or BRCA2 mutations, are tested negative for the same BR CA1 and BRCA2 mutations, those relatives are still at increased risk of cancer  (2) Positive ("Positive for a deleterious mutation" or "Suspected delete rious"). In this case, the risks for the individual to devel op breast and ovarian cancer are much hig h er than the risk for the general population (Table1). (3) Uncertain ("Genetic change of uncert ain significance"). This result means a genetic change in BRCA1 for which the impact on protein function has not been dete r mined. These genetic changes are cal led unclassified variants or genetic variants of u n certain clinical significance (VUS s ). It is es timated that around 10% of Caucasians, 35% of Hispanics, and up to 50% of African America ns undergoing testing receive VUS r e sults [71 73] Considering over 1500 different alleles of BRCA1 in the human population (B reast cancer information core http://research.nhgri.nih.gov/projects/bic as of 3.9.11 ) and the rarity of the individual alleles, one of the most challenging tasks for clinical g e netics is to distin guish which are benign and which are cancer predisposing. These three outcomes are similar for BRCA2 testing. How to Determine the Association of a Genetic Variant with a D isease? There are two methods that can be used for determining association of a g enetic variant with a disease (cancer). The first method is defined as genetic i.e. test ing co
13 segregation between the variant and the disease in families in which this variant has been found. The second method is epidemiological i.e. comparing the freque ncy of a given variant between two groups. These groups are cases (individuals from different families that have the disease) and controls  In the cas e of BRCA1 VUS s both methods have limitations. The genetic method cannot always be applied because usually the families are small, the number of genotyped family members is limited, and there is an uncertainty about which type of cancer (diagnosis, histo logy, ext.) a deceased or distant family me m bers present ed The epidemiological method relies on relatively common alleles but i n dividual VUS are very rare in the population. To overcome this problem, several methods to help classify the VUSs have been pr oposed: 1. Functional assays to test the effect of amino ac id change on the function of BRCA1: 1.1 Transcription activation assay [75, 76] and reviewed in  BRCA1 C ter minus functions as an activator of transcription [78, 79] when expressed as a fusion with the heterologous DNA binding domain. Cancer predi s posing mutations in this region of BRCA1 impair this transcriptional activi ty whereas benign polymorphisms show similar activity as the wild type BRCA1 [80, 81] This fact served as the basis for the development of the transcription a c tivation assay, whi ch can test the integrity of BRCT d omain s and its neighboring regions at the BRCA1 C terminus. This assay is limited to the C terminal mutants of BRCA1.
14 1.2 Yeast small colony phenotype assay [82, 83] Yeast small colony ph enotype is based on the premise t hat constructs, which co n tain the C terminus of BRCA1, inhibit the yeast growth when expressed. This a s say is limited to the C terminus of BRCA1. 1.3 E3 ubiquitin ligase activity assay  The BRCA1/BARD1 heterodimer displays a n E3 ubiquitin ligase activity  Therefore, functional assays have been developed to test BRCA1 variants for their ability to bind to BARD1 and UBCH5A (the E 2 ubiquitin conjugating enzyme). In addition, such functional assays test BRCA1 in vitro E3 ligase activity. This assay is domain specific and can test variants only in the N terminus of BRCA1. 1.4 Phosphopeptide binding assay Phosphopeptide binding assay evaluates the ability of BRCT domain s to interact with a phosphopeptide from BACH1 pr otein This assay is also limited to the BRCT domains of BRCA1  1.5 Protease sensitivity assay Protease sensitivity assay tests for the stability of BRCT domains upon cleavage with protease. Some subtle deleterious changes i n BRCA1 may not be detected when using this assay  1.6 Yeast recom bination assay Yeast rec ombination assay tests for BRCA1 C terminu s variants effect on the recombination between the yeast HIS3 and ADE2 loci. This type of assay is r e stricted to the BRCT domain s 
15 1.7 Human BRCA1 BAC reconstitution into mouse embryonic stem cells which have conditional Brca1 allele, follow ed by functional tests for cell viabil i ty, DNA repair, and m ammary gland carcinogenesis  The same approach can be used to rescue embryonic lethality into nullizygous for the mouse Brca1 allele embryos  2. Co occurrence in trans with a deleterious mutation This method is based on the observation that some functions of BRCA1 are essential for early development. For example, homozygous truncating mutations in BRCA1 are embryonic lethal in mice [45, 46] Moreov er, so far no patient has been reported ha v ing two deleterious mutations in BRCA1 gene  Thus, this method is based on the hypothesis that homozygosity for deleterious mutations is either embryonic l e thal or lead to recognizable phenotype  If a certain VUS of BRCA1 co occurs with a known deleterious mutation in one tested individual most likely this VUS is a benign polymorphism. 3. Bioinformatics methods is a structure based analysis, applied to generate computation pr ediction models [91, 92] and analysis of BRCA1 sequence conse r vation among different species and the amino acid characteristics [93 95] All the methods mentioned above have their own assumptions, ad vantages, and disadvantages. Therefore none of these methods should be used alone to determine the dise ase association of VUS s or as a clinical application. In addition, several attempts have been made to combine different sources of data to classify VUS [91, 95 97] Even
16 after using multiple sources of data the classification of VUS s is far from being directly applied in the clinic, which is the ultimate goal of any research on the VUS s of BRCA1 To address this problem a nd to improve the clarity in conveying the information from genetic testing to the patients, the scientific community recommends the usage of a five scale classification, which includes the information about how relevant th e particular BRCA1 variant is for the clinical practice  According to this scale the results from the genetic testing for BRCA1 and BRCA2 mutations can fall into one of the five categ o ries  Class 1 c orresponds to variant to be pathogenic in this category is below 0.1%. Class 2 c Likelihood for a variant to be pathogenic in this category is between 0.1 and 5%. Class 3 c Likelihood for a variant to be pathogenic in this category is between 5 and 95%. Class 4 c variant to be pathogenic in this category is between 95 and 99%. Class 5 c variant to be pathogenic in this category is above 99%. Each of t hese classes is accompanied by recommendations for treatment and surveillance options. As more patients are tested and the methods for classification are imp roving fewer variants will fall into C lass 3  Therefore the results from the
17 functional assays ca n be a valuable addition for clinical geneticist s when advising their p a tients. Importantly, the accumulated knowledge on functional assays to classify alleles of BRCA1 and BRCA2 can be applied to any other disease predisposing gene. In summary, information for BRCA1 or BRCA2 status is the most predictive factor for the risk of breast and ovarian cancer development. The increase in risk is substantial and is almost five to ten times higher than the risk in the general population. Moreover, deleterious mutations in the BRCA1 gene have high penetrance r e sponse to treat ment is highly influenced by their BRCA1 status However, despite the enormous efforts to classify VUS s and to help patients with their clinical decisions, our ability to identify at risk women remains problematic One of the main limitati ons is the lack o f knowledge about mechanistic aspects of BRCA1 biology. BRCA1 is a prot ein with pleiotropic functions and it is not known how many of those functions alone or in combination contribute to the functions of BRCA1 as a tumor suppressor. To bridge the gap bet ween the lack of understanding of BRCA1 role in tumor suppression on one hand and the ability to classify the BRCA1 VUS s on the other, we set out to systematically evaluate the biochemical function of BRCA1 do mains and motifs. In this dissertation we focu s on analyzing the function of two BRCA1 conserved motifs called Motif 2 and M o tif 6.
18 Why Are Studies on BRCA1 I mportant? Mutations in BRCA1 and BRCA2 are responsible for 5 10% of all breast cancer cases. A common critique to studies on BRCA1 is that a relatively small number of wo m en with inherited mutations of BRCA1 limit their impact on the disease burden. Then, why should we study BRCA1 ? To summarize the studies on BRCA1 are important for the patients that carry deleterious mutations in BRCA1 and their families because of the following reasons : 1. Early detection of individuals at risk is important so that they and their families can make informed clinical decisions about prophylactic surgery, increased su r veillance, chemoprevention, and risk for othe r malignancies. The presence of harmful BRCA1 mutation in a family influences other decisions in life such as childbearing. 2. Individuals that have already developed breast cancer and have deleterious mutation in BRCA1 will respond differently to radiotherap y and chemotherapy than the individuals from the general population. Moreover, studies on BRCA1 have much broader implications, because: 1. Approximately 10% of all genetic tests find VUS (this percentage is higher in some populations see page 2 ), which t ranslates into the fact that ~ 10 000 families in the US alone, cannot make informed clinical decisions. 2. BRCA1 is epigenetically silenced in sporadic tumors [27, 32] and may regulate mammar y stem/progenitor cell fate [99, 100]
19 3. Many genes implicated in breast cancer function in DNA damage response pathway s. Thus, an understanding of BRCA1 role will likely have an impact on other forms of breast cancer not attributable to germline mutations in BRCA1. In addition, ot h er dise ases such as Fanconi Anemia arise from defects in the BRCA1 related pathways. 4. Radiation therapy and many chemotherapy drugs act by causing DNA damage in the cells. BRCA1 is a n important participant in the cellular response to DNA damag e, which makes it very essential factor in many cancers including l ung and ovarian cancers [101, 102] The DNA Damage Response (D DR) Network DDR: DNA Repair and Cell Cycle Checkpoints In order to preserve genome integrity different evolutionarily conserved DNA cellular processes such as the DNA damage response (DDR) and cell cycle checkpoints have evolved  The DDR may be defined as a collective name for the processes with the help of which cells detect DNA damage, initiat e DNA repair, and coordinate r e pair with cell cycle progression. Therefore, the DDR is a complex network of different interco nn ected pathways. W e arbitrarily divide the protein complexes involved in the DDR as sensors, signal transducers, effector kinases, mediators and effector proteins [3, 104] C ell cycle checkpoints represent the mec hanisms by wh ich cells stop (during G1,
20 G2 phases) or slow down (during S phase) cell cycle progression until an earlier process such as replication or mitosis is completed  After DNA repair is accom plished, cells either resume cell cycle progression or, if the damage cannot b e repaired, perm a nently arrest, s enesc ence, or die (apoptosis)  Mutations in ATM, TP53, NBS1, BRCA1, BRCA2, CHEK2, PALB2, BACH1, and other genes lead to an increased risk for the developme nt of breast cancer if compared to the risk of the general population  These proteins participate in the DDR network. NBS1, p53, BRCA1, BRCA2, CHK2, and ATM itself are substrates for the ATM kinase activity, when the cells experience DNA damage (Figure 2) Moreover, cells, lacking full length functional ATM, NBS1, and BRCA1, share similar phenotypes such as cell cycle checkpoin t defects, radio sensitivity, increased chromosomal breakage, and failure to phosphorylate SMC1 (cohesin subunit) after IR  It is currently unclear why mut a tions of genes, the products of which are operating i n a conserved mechanism active in e very cell, lead primarily to breast and/or ovarian cancer predisposition. Double strand break ( DSB ) repair can be achieved mainly by two mechanisms: non homologous end joining (NHEJ) and homologous recombination (HR) [106, 108] and ( Figure 3 ) On one hand, NHE J is dependent on DNA PKcs, Ku86 Ku70, and DNA ligase IV  It can be perform ed throughout all cell cycle stages and is error prone. In addition to NH EJ, there is a less studied Ku86 and DNA Ligase IV independent altern a tive end joining pathway  On the other hand, DSB generated during S and
21 Figure 2. Breast cancer susceptibility gene products and the DDR pathway. In this simplified view ATM is activated by the presence of DSB and phosphorylate CHEK2, BRCA1, and TP53. Activated CHEK2 also phosphorylates TP53 and BRCA1. Note that BRCA1 and NBS 1 contribute to the ATM activation and can function both upstream and downstream of ATM. Phosphorylation of these proteins is required for the efficient activation of various cell cycle checkpoints. BRCA2 regulates the function of the human recombinase RAD 51. PALB2 act as a bridge b e tween BRCA1 and BRCA2. Another protein implicated in breast cancer predispos i tion, PTEN, mediates down regulation of AKT. (Adapted from  ).
22 Figure 3. DNA repair by homologous recombination and non homologous end joining. The main steps of the two repair mechanisms are shown. (Adapted from  ).
23 G2 phases of the cell cycle are predominantly repaired by HR, because sister chromatids are available to serve as repair templates. HR requ ires processing of DSB to generate si n gle stranded DNA (ssDNA). ssDNA fulfills two roles: activates the checkpoint kinase ATR and is the only substrate that allows Rad51 recombinase to be loaded onto chrom a tin. HR is an error free repair pathway  Every day our cells are exposed to different DNA damaging agents that originate from their own metabolism (reactive oxygen species) or from their surroundings ( ultr a violet ( UV ) radiation IR, and different drugs)  Different agents cause different types of DNA damage  In this dissertation we focus on DNA damage caused by i o nizing radiation (IR), which primarily leads to double s trand breaks (DSB)  In some cases, we use hydroxyurea (HU) and campthothecin (CPT). To cause DNA damage, HU blocks DNA replication by inhibiting ribonucleotide reductase. Further, stalled replic a tion forks undergo irreversible collapse and/or are processed to DSB  Another e f fect is caused by CPT, which is a T opoisomerase I inhibitor that generates replication mediated DSB  In the current study, we investigate the extent to which different signal tra nsduction pathways are activated after treating the cells with IR, HU, or CPT. Sensors, Signal Transducing Kinases, Effector Kinases and Effector Proteins Upon DNA damage, three signal transducing Ser/Thr kinases: ATM, ATR, and DNA PKcs are rapidly activated and recruited to the damaged areas in the DNA 
24 ATM and DNA PKcs are mainly activated in response to DSB, whereas ATR is activated by ssDNA and stalled replication forks  which consequently generate ssDNA, coated with RPA  Onc e at the proper place, the kinases relay the D NA damage si g nals by phosphorylating numerous downstream substrates. For example, ATR and ATM directly phosphorylate and activate the main effector kinases CHK1 and CHK2. Act i vated CHK1 and CHK2 kinases then regulate the function of effector proteins such as p53, E2F, and Cdc25 phosphatases, which in turn directly regulate cell cycle progression  When DNA dam age occurs, DSB are first recognized by the MRN (Mre11/Rad50 /Nbs1) complex and the Ku70/Ku86 heterodimer  which are all considered sensors for DSB. In addit ion, the ssDNA and stalled replication forks are reco g nized by Rad9/Rad1/Hus1 complex (also known as the 911 complex) and the ATRIP/ATR complex  It has been shown that conserved mot ifs in NBS1, Ku86 and ATRIP interact with and are responsible for chromatin loading of ATM, DNA PKcs, and ATR respectively  Even though ATM and ATR overlap in the phosphorylation of t heir substrates, there are differences in their relative contribution. This contribution depends on the type of the genotoxic stress  For instance, cells that over express a kinase dead mutant of ATR are hypersensitive to UV, HU, and IR, while ATM deficient cells are hypers e n sitive only to IR  According to current models, it is accepted that ATM and ATR are activated by different DNA structures, rather than specific genotoxins  A dding complexity to the DDR, these various DNA structures can be converted to one
25 another. For example, DSBs, generated after IR will primarily activate ATM. How ever, different enzymatic activities (nucleases, helicases, etc.) during the end processing of DSB after IR often generate ssDNA, coated with RPA which subsequently activates ATR  Fu r thermore, ATR is importan t for maintaining genomic integrity not only after genotoxic stress but also during normal cell cycle progression. Checkpoint Mediators Mediators in the DDR are proteins that act directly downstream of the ATM and ATR kinases  such as MDC1, 53BP1, BRCA1, the MRN complex, Claspin, and MCPH1. Mediators have different roles in the DDR e.g. control over localization activ a tion of other factors and act as a scaffold for the assembly of protein complexes [118, 119] As an example, we will briefly describe the signaling events that lead t o the recruitment of BRCA1 to the sites of DSB after DNA damage (Figure 4) There, once r e cruited BRCA1 can be visualized as discernible nuclear foci called irradiation induced nuclear foci (IRIF) [120 123] Firs t, after the DNA damage occurs, one of the earliest targets of activated ATM, ATR, and DNA PKcs is the histone H2A variant H2AX  It is phosphorylated at a conserved Ser 139 residue on both sides of the DSB, cr eating a specialized chromatin domain  H2AX. Because of H2AX is the most widely used DSB marker in western blots and in i mmunofluorescence analysis of foci formation. Our own work, which is not
26 Figure 4. Current model for the BRCA1 recruitment to the IRIF. DSBs are re c ognized by the MRN complex, which recruits activated ATM to the DNA lesion. ATM phosphorylates H2AX (for simplicity nucleosomes are depicted as blue ci r cles). MDC1 binds directly to the phosphorylated H2AX and recruits the ubiquitin ligase RNF8. RNF8 initiates histone ubiquitylation at the damaged sites. This chr o matin modification allows the recruitment of BRCA1 to the sites of DSBs.
27 included in this dissertation, demonstrated the importance of H2AX post translational modifications in the regulation of its own turnover, cell cycle, and apoptosis  After the phosphorylation of H2AX, MDC1 is recruited to the DSBs. MDC1 possesses tandem H2AX  The major function of M DC1 is to amplify H2AX phosphorylation by recruiting additional ATM molecules or H2AX dephosphorylation  Such amplification allows for the recruitment of additional DDR factors to the sites of damage, most of which ca n be visu a lized as IRIF  According to the current understanding, the n ext event in the recruitment at the sites of DNA damage is the interaction between the FHA domain of the E3 ubiquitin l i gase RNF8 and phosphorylated MDC1 [129 131] (Figure 4) Once recruited to the DSB, RNF8 catalys es Lys 63 linked ubiquitination of histones and other proteins   H2AX stabilizes the recruitment of 53BP1 and BRCA1 at the DSB sites  For example, a protein called Rap 80 possesses both a ubiquitin interac t ing motif and a coiled coil domain. The Lys 63 ubiquitinated histones are recognized by the ubiquitin interacting motif of Rap80   Furthermore, the coiled coil domain of Rap80 directly interacts with the coiled coil domain of Abraxas  The recruitment of BRCA1 at the sites of DSB is mediated by the binding of its BRCT domains to the phosphorylated Abraxas   (Figure 4) Once in the proper location at DSB chromatin, BRCA1 coordinates DNA repair, transcription, and cell cycle checkpoints by facilitating the phosphorylation of downstream substrates such as p53, NBS1, CHK1, and CHK2 in an ATM/ATR dependent manner  The mechanistic
28 details of how BRCA1 modulates the phosphorylation of such a large number of substrates are still u n clear. Moreover, ATM and ATR kinases phosphor ylate BRCA1 at the numerous sites thus modulating BRCA1 activity. Further, BRCA1 interacts with ATR partner ATRIP  promoting ATR dependent checkpoint functions. However, the mechanism d e scribed above is not the only one that leads to the recruitment of BRCA1 to the DSBs sites. Even in the absence of H2AX /MDC1/Rap80/Abraxas mechanism, BRCA1 still localizes to the sites of DSBs  Finally, BRCA1 acts as a scaffold for the assembly of different complexes after DNA damage. It has been demonstrated that three major BRCA1 containing complexes are formed after IR: one that contains BACH1 and is involved in the intra S phase checkpoint, another CtIP and MRN complex that participates in the early G2/M chec k point, and the third BRCA2/Rad51 that is responsible fo r DNA repair by HR  DDR: Spatiotemporal Regulation in Specialized Chromatin Domains In addition to the widely used classification of DDR participants such as sensors, mediators etc., Bekker Jensen and collea gues divide DNA repair complexes into three groups, depending on their location related to the DSB: DSB flanking chromatin (chr o matin compartment), single stranded DNA micro compartment, and repair complexes that do not show retention at the DSB  DSB flanking chromatin is occupied by groups of proteins that assemble on both sides of the DSB and can spread megabases from both sides of the break  Proteins that locali ze to this compartment
29 are ATM, MRN complex, MDC1, 53BP1, and BRCA1. In addition, single stranded DNA micro compartment is occupied by the proteins such as the single strand DNA binding protein RPA, ATR, Rad51, BRCA2, MRN complex, and BRCA1. Finally, compl exes that do not show retention at the DSB are DNA PKcs, Ku70, Chk1, Chk2, and p53. These specialized chromatin domains perform different functions in DNA repair and checkpoint signaling. Importantly, the proteins that occupy the DSB flanking chr o matin ca n assemble during all cell cycle stages. Meanwhile, complexes that occupy the single stranded micro compartment assemble in a cell cycle dependent manner only du r ing S and G2 phases. MRN complex and BRCA1 have unique roles in DDR signaling and processing o f DSB because they are the only proteins that can be found in two co m partments (double and single st randed). This fact is essential when discussing dependence in the recruitment of different proteins to the DSB sites and their functional cons e quences. For example, the down regulation of MDC1 leads to the dissociation of BRCA1 only in the chromatin, but not in the single stranded compartment  On the contrary, the down regulation of BRCA1 leads to the dissociatio n of BRCA2/Rad51 complex from the single strand compartment, showing that BRCA1 is upstream of BRCA2/Rad51 in the DNA repair signaling. In addition, it has been proposed that different protein complexes contribute to the recruitment of BRCA1 to the differe nt compartments  For i n stance, MDC1/ Rap80/Abraxas complex localizes BRCA1 to the chromatin compartment (see the previous section), and MRN complex and CtIP to the single stranded DNA m i cro compartment. Most likely the disruption of BRCA1
30 function in these different loc a tions will have different functional consequences for the DNA repair and checkpoint si g naling. BRCA1 : Structure and Functions The Structure of BRCA1 The BRCA1 gene locus is located on c hromo some 17q21 and codes for a 1863 amino acid s protein  The conserved domains and motif s of BRCA1 are shown on Figure 5 and summarized in Table 3. At its N terminus, BRCA1 has a zinc binding RING finger domain (aa 1 101)  In vivo, most of BRCA1 resides in a complex with BARD1, which also contains a RING finger domain [139, 140] BRCA1 and BARD1 interact with each other via their respective RING finger domains and form a stable het e rodimer  RING finger domains possess intrinsic E3 ubiquitin ligas e activity  Consistent with this, BRCA1/BARD1 complex has E3 ubiquitin ligase activity  [142, 143] Within the BRCA1 RING domain, cancer causing mutations lead to the abrog a tion of BRCA1 E3 ubiquitin lig ase activity and fail to restore radiation sensitivity of a BRCA1 deficient cell line  Moreover, BRCA1 variants at the N terminus of BRCA1, which dis rupt BRCA1 binding to the E2 enzyme, lead to the loss of ubiquitin ligase activity  Thus, loss of ubiquitin ligase activity correlates with susceptibility to breast and ovarian cancer 
31 Table 3. BRCA1 domains and motifs Domains and M otifs Amino acids Function Refs RING 1 101 E3 ubiquitin ligase  NES 81 89 Nuclear export  Mot if 1 123 130 u nknown  Motif 2 178 189 u nknown (studied in this thesis) [145, 146] Ser 308 308 Aurora A phosphorylation targe t site  Motif 3 378 388 unknown  Motif 4 458 467 unknown  DNA binding region 452 1079 Binding to branched DNA  NLS 503 508, 651 656 Nuclear import  Motif 5 512 521 unknown  Motif 6 845 869 u nknown (studied in this thesis) [145, 146] Ser 988 988 CHK2 phosphorylation target site  Motif 7 1147 1153 unknown  Ser 1189 1189 Cdk1 phosphorylation target site  Ser 1191 1191 Cdk1 phosphorylation target site  Motif 8 1208 1228 unknown  Coiled coil 1369 1418 PALB 2 binding  Ser 1387 1387 ATM/ATR phosphorylation target site, intra S phase ch eckpoint  Ser 1423 1423 ATM/ATR phosphorylation target site, G2/M checkpoint  Ser 1497 1497 Cdk1/Cdk2 phosphorylation target site, [151, 153] Ser1524 1524 ATM/ATR phosphorylation target s ite,  Ser1572 1572 CK 2 phosphorylation target site  BRCT1 [N] 1650 1753 DNA damage signaling, transcription  BRCT2 [C] 1760 1855 DNA damage signaling, transcription 
32 Figure 5. Structure of BRCA1 RING and BRCT domains are shown in red boxes; Coiled coil domain is depicted as a black box; NLS, nuclear localization signal;M2, Motif 2; M6, Motif 6; The regions of BRCA1 that have evolutionary conserva tion above 75% are shown on the blue scale bar (Adapted from  ).
33 At its C terminus, BRCA1 has two BRCT domains in tandem  The BRCT domain was first described in BRCA1 (BRCT stands for BRCA1 C terminal domain) and was found in several proteins such as MDC1, p53BP1, and DNA ligase IV, which all pa r ticipate in DNA repair and checkpoint control   It has been shown that tandem BRCT domains bind phospho peptides [158, 159] In particular, the two BRCT domai ns of BRCA1 behave as one functional unit that binds to phosphorylated proteins with the consensus sequence pSer X X Phe (where X is any amino acid)  Moreover, BRCT domains are important for the functions of B RCA1 as a tumor suppressor. According to the genetic evidence, all mutations found in cancer patients that lead to the truncation of BRCA1 BRCT domains confer a high risk for cancer development  Thus, such m u tations are currently classified as deleterious variants  The remaining central region of BRCA1 between the RING and BRCT domains has not been studied in depth. However, this region of BRCA1 contains several signif i cant features. They include nuclear localization (aa 503 508), nuclear export sequences (aa 81 99) [144, 162] the coiled coil domain, and BRCA1 aa 452 1079 region, important for binding to branched DNA structures  The coiled coil domain encompasses BRCA1 exons 12 and 13 (aa 1391 1424) ( see 5 ) [94, 163] It has bee n found to interact with two proteins: JunB  and PALB2 [164, 165] Importantly, mutations within the coiled coil domain found in breast and o varian cancer patients modulate the ability of BRCA1 to activate transcription  Moreover, mutations such as L1407P and M1411T mapped to the BRCA1 coiled coil domain disrupt the binding
34 between BRCA1 and PALB2 and are defective in homologous recombination repair assays  The ev i dence described above suggests a potential role of the coiled coil domain in tumor su p pression. Furthermore, the biological role of some BRCA1 phosphorylation sites has already been studied. For example, BRCA1 double mutant Ser 1423Ala/Ser 1524Ala ca n not rescue the radiation sensitivity of BRCA1 deficient cells, showing that th ese two sites are important for survival after IR  In addition, phosphorylation of Ser 1423 is r e quired for the IR induced early G2/M checkpoint  and Ser 1387 for the intra S phase checkpoint activation after IR  Finally, phosphorylation of Ser 988 was r e ported as important for the role of BRCA1 in homologous recombination repair  and in mitosis  Importantly, only one of these sites, Ser 1524, has been found mutated in patients with breast and ovarian cancer. However, its association with cancer has not been determined  Finally, nine additional conserved segments   were revealed during the analysis at the conserved positions in BRCA1 orthologs. Such segments are called Motifs 1 to 8 and the coiled coil domain of BRCA1 is referred to as Motif 9. The contribution of these nine motifs to different BRCA1 fu nctions is still u nder researched. Analyzing the function Motif 2 and Motif 6 is the main focus of this dissertation.
35 BRCA1: Biochemical Activities and Biological P rocesses BRCA1 participates in many biochemical activities and biological processes and thus has a plei otropic role in the cell. In fact, there is often a confusion between First, BRCA1 participates in such central processes in the life of a cell as DNA repair, control of the cell cycle, transcriptio n, and mitosis  ( Figure 6 ) In addition, many biochemical activities ascribed to BRCA1 including transcription activation and repression [79, 170] chromatin remodelin g  and ubiquitination  However, it is not clear, which of these functions and activitie s, alone or in a combination, contribute to BRCA1 role as a tumor suppressor. Though many of these BRCA1 functions and activ i ties are not tissue specific and BRCA1 is a ubiquitously expressed protein, it is not clear why individuals heterozygous for BRCA1 mutations develop mainly breast and/or ov a rian cancer. Thus, understanding of BRCA1 biology is ultimately linked to our ability to provide risk assessment and better treatment options for the patients with BRCA1 associated breast and ovarian cancer. Seco nd, research on BRCA1 functions has shown that BRCA1 activities in the cell are regulated by different post translational modifications such as phosphorylation  ubiquitination  sumoylation [173, 174] and interactions with close to 100 different cellular proteins. Another level of such regulati on happens when BRCA1 e x pression is tightly controlled during cell cycle progression. For example, BRCA1 protein level is low in G0 and G1 phases of the cell cycle and increases as cells enter S
36 phase [149, 175] Fu rthermore, BRCA1 localizes to discrete nuclear foci (dots) during normal S phase, but it re locates to PCNA containing replication foci after DNA damage  Finally, BRCA1 participates in many aspects of DNA dama ge signaling pathways such as intra S phase checkpoint [107, 166]  G2/M checkpoint   spindle assembly checkpoint [169, 177] end processing [178, 179] and DNA repair both by HR and NHEJ (Figures 3, 6 and Table 4 ) In this dissert ation we focus on the functions of BRCA1 in the DDR. G1 C heckpoint The signal transduction pathway that is responsible for the cell cycle arrest in the G1 stage of the cell cycle includes ATM, ATR, CHK2, CHK1, MDM2, TP53, and p21  The ultimate goal of this pathway is to stop the cell cycle by increasing the stabil i ty and transcriptional activity of TP53 protein  TP5 3 controls the expression of CDK inhibitor p21, which in turn inhi bits the kinase activity of cyclin E/Cdk2  The main mediators of G1 checkpoints are the tumor suppressors TP53 and pRB. BRCA1 has been implicated in interactions with both p53 and pRB and it is thought that BRCA1 pa r tic ipates in G1 checkpoint control by its numerous interactions with ATM, ATR, RB, p53 and p21   However, the molecular details and the end point effects of BRCA1 deficiency with regard to G1 checkpoint are not clear
37 Intra S Phase Checkpoint The intra S  For example, it can be triggered by the IR induced DSBs. The activation of intra S phase checkpoint leads to a rapid decrease in DNA synthesis after DNA damage b e cause of the inhibition of new origin firing  Cells with defective intra S phase checkpoint control fail to down regulate DNA synthesis and display a radio resistant DNA synthesis (RDS) phenotype [115, 182] The RDS pheno type has been observed in cells deficient in p roteins such as: ATM, ATR, CHK1, CHK2, NBS1, 53BP1, BRCA1, and BRCA2  This suggests these proteins' par ticip ation in the activation or mai n tenance of the checkpoint. Finally, it has been demonstrated that the intra S phase chec k point is cooperatively controlled by at least two parallel ATM dependent mechanisms: the ATM NBS1 BRCA1 SMC1 [107, 182] axis and ATM CHK2 CDC25A Cdk2 axis  and ( Figure 7 ) In the first pathway, ATM dependent phosphorylations of SMC1 at Ser957 and 966, NBS1 at Ser 343, and BRCA1 at Ser1387 are necessary for the intra S phase chec k point activation [152, 166] However, it is not clear how phosphorylated SMC1 leads to down regulation in DNA replication. In addition, the functional consequences of NBS1 and BRCA1 phosphorylations are poorly understood. To complicate this matter
38 Figure 6. Cell cycle stages and participation of BRCA1 in cell cycle checkpoints. G1, gap phase 1;G2, gap phase 2; S, DNA synthesis stage; M, mitosis. Participation of BRCA1 in different cell cycle checkpoints is shown with red arrows. (Adapted from http://homepage.mac.com/enognog/checkpoint.htm ).
39 Figure 7. The intra S phase checkpoint signaling. The two parallel pathways that coop e ratively control this checkpoint are depicted. ( Adapted from  ).
40 further, it has been demonstrated that the phosphorylations of BRCA1 at Ser1387 and NBS1 at Ser343 are not necessary for the ATM dependent phosphorylation of SMC1 at the Ser 957 and 966 sites after IR. Instead, it has been hypothesized tha t BRC A1 and NBS1 might be needed as scaffold proteins in order for SMC1 phosphorylation to occur properly after DNA damage  According to the current understanding, the recruitment of BRCA1 and NBS1 to the DSB s happens independently of ATM phosphorylation. Moreover, the initial activ a tion of ATM occurs independently from BRCA1 and NBS1  However, BRCA1 and NBS1 are required for the proper localization of once activa ted ATM to the break sites  Only then, ATM c an phosphorylate substrates such as BRCA1, NBS1, and SMC1. Moreover, BRCA1 and NBS1 phosphorylations are dependent on each other and on the proper ATM recruitment to the DNA DSB sites  The other signaling pathway implicated in the intra S phase checkpoint is ATM CHK2 CDC25A Cdk2  Signaling through this pathway leads to Cdc45 inability of being loaded at the replication origins. Howev er, such loading is necessary for the initi a tion of DNA replication  Interestingly, BRCA1 interacts with BACH 1 helicase and TopBP1. BRCA1, BACH1, and TopBP1 appear to control Cdc45 origin licensing factor after DNA damage through poorly understood mechanisms  Thus, in addition to ATM NBS1 BRCA1 SMC1 pathway, BRCA1 might also participate in the ATM CHK2 CDC25A Cdk2 axis of the intra S phase checkpoint by controlling Cdc45. In conclusion, ATM initiates two parallel pathways th at are responsible for the a c tivation of intra S phase checkpoint by phosphorylating proteins such as CHK2,
41 BRCA1, NBS1, and SMC1. The examples discussed above illustrate the importance of BRCA1 in the regulation of different steps in DDR: functioning both upstream and downstream of ATM and acting as a scaffold for the assembly of multiple protein complexes. In addition to BRCA1, ATM, and NBS1, and another BRCT domain containing protein DNA re p lication factor C has been purified as a part of a large super complex named BASC (BRCA1 associated genome surveillance complex)  This shows a potential link b e tween BRCA1 and replication associated repair and signaling. Clearly, more research is necessary to elucidate m echanisms behind the complex role of BRCA1 in the intra S phase checkpoint. Any molecular details on this process will likely translate not only into better surveillance options for the patients with harmful BRCA1 mutations, but also into designing improv ed treatments for cancer. G2/M C heckpoint ATM CHK2 and ATR CHK1 pathways control the checkpoint responses in the G2 phase of the cell cycle by phosphorylating the mitosis promot ing phosphatase CDC25C (Figure 8 ). This phosphorylation inhibits the CDC25C, and it cannot depho s phorylate and thus activate cyclin B/CDK1 kinase complex  Consequently, these events block the cells in G2/M phase of the cell cycle. Moreover, the maintenance of the G2/M checkpoint relies on the transcription initiated by p53 and BRCA1. Thus,
42 Figure 8. The G2/M checkpoint signaling. Main sensor (911 complex), med iators (MDC1, BRCA1, and 53BP1), effector kinases (CHK1 and CHK2), and effector protein (Cdc25 phosphatase) are shown. (Adapted from  ).
43 BRCA1 participates in G2/M checkpoint signaling via the transcriptional regulation (both activation and repression) of genes responsible for G2/M checkpoint control  These genes include Cyclin B, 14 3 3 proteins, the Wee 1 kinase, and p21  Finally, BRCA1 regulates the transcription of GADD45a  which in its turn regu lates cyclin B/CDK1 kinase complex by the inhibition of its kinase activity  However, besides its role in transcriptio n, BRCA1 participates in the G2 phase checkpoint responses by being a target of ATM and ATR kinases. They both phosphor y late BRCA1 on multiple residues (see Table 2). According to Kastan and colleagues, there are two distinct G2 checkpoints: early G2/M checkpoint and G2 accumulation which are measured by different methods and at different time points after IR  I m portantly, both checkpoints require BRCA1 participation [152, 188] In particular the expression of BRCA1 S1423A mutant in a BRCA1 deficient breast cancer cell line HCC1937 can reconstitut e early G2/M checkpoint, but not G2 accumulation  This indicates that the two checkpoints are controlled by different signaling events. The early G2/M checkpoint  occurs early after IR (1 2h) and is ATM d e pendent. Furthermore, it occurs in cell s in the G2/M boundary at the time of IR that fail to progress to mitosis  On the contrary, the G2 accumulation checkpoint is dose dependent and ATM independent  It measures the cells that have been in S and G1 stages at the time of irradiati G2 stage  The G2 accumulation checkpoint is usually measured at a later time after IR (i.e. 24h). It is important to point out, that BRCA1 or NBS1 deficient cells, that have
44 a defect in IR induced intra S phase checkpoint, usually display G2 accumulation phen o type  Another checkpoint, called the Human decatenation checkpoint, can be related to the G2 checkpoint responses in the cell, although it is not a response to IR. The H u man dec atenation checkpoint monitors chromatid decatenation in cells that progress from the G2 phase of the cell cycle to mitosis  If sister chromatids are insufficiently decatenated, the progression from G2 phase to mitosis is blocked  In particular, ATR and BRCA1 work together to control G2 decatenation checkpoint by excluding cyclin B1/Cdk1 complexes from the nucleus  On t he molecular level, BRCA1 ubiquitinates topoisomerase II alpha and regulates its decatenation activity [1 90] Finally, H u man decatenation checkpoint seems to be independent of ATM  Importantly, failure in any given checkpoint by itself does not lead to radio sens i tivity  This can be exemplified by BRCA1 m utants, expression of which affect irradiation [152, 166, 188] For example, the expression of BRCA1 S1387A mutant reverses the ea r ly G2/M checkpoint defect but not the intra S phase checkpoint defect [152, 166] On the contrary, S1423A mutant reverses the intra S checkpoint defect but not the early G2/M phase checkpoint defect  Therefore, radiation sensitivity as a phenotype is not ne c essarily a consequence of the defective checkpoint signaling; rather it represents a more complex phenotype because of the failure in more than one stage in the DDR. Thus, the fact that cell lines that do not express functional BRCA1 have
4 5 severely compromised long term survival after irradiation illustrates that BRCA1 functions on several sta ges in the DDR and performs multiple functions after IR. Spindle Assembly C heckpoint The spindle assembly checkpoint monitors the accuracy of chromosome segreg a tion by preventing cells with misaligned chromosomes from exiting mitosis [180, 191] BRCA1 regulates the transcription of several spindle assembly checkpoint genes (inclu d ing Mad2) [177, 180] Mouse embryonic fibroblasts isolated from Brca1 mice di s played chr omosomal abnormalities (bridges and lagging chromosomes), which are hal l marks of spindle assembly checkpoint defects  The s tudies of spindle assembly checkpoint related to BRCA1 in m ammalian cells have been difficult because the inhib i tion or over expression of BRCA1 lead s to cell cycle arrest or apoptosis  The d own regulation of BRCA1 in human prostate cell line DU 145 (which e x press es wild type BRCA1) and human breast cancer cell line MCF7 (hemizygous for wild type BRCA1 expression) followed by microarray analysis revealed that BRCA1 control s the transcription of many genes that are required for mitotic progression  Moreover, it has been demonstrated that checkpoint kinase Chk2 and its target BRCA1 (phosphorylated at Ser 988 ) function independently of p53 and DNA damage in mitosis. Chk2 kinase and BRCA1 are required for normal progression of mitosis and subsequen t ly maintaining chromosomal stability 
46 DNA R ep air by HR HR is an error free DNA repair mechanism, wh ich uses the sister chromatid or less often the homologous chromosome as a repair template  and ( Figure 3 ) BRCA1 participates in the regulation of HR. The research on such regulation involved the measurement of HR efficiency in BRCA1 deficient cell lines [194, 195] association of BRCA1 with other proteins participating in different stages of HR (see below) and the decrease of Rad51 foci formation i n the absence of functional BRCA 1 [121, 137, 196] As an example, mouse cell lines, whic h do not express functional Brca 1, have decreased HR efficiency and increased genomic instability [194, 195] In addition, several BRCA1 interacting partners participate in the control of HR repair. These include a growing nu m ber of proteins such as BRCA2  PAL B2  BACH1  CtIP [178, 198] 53BP1 an d the MRN protein complex [3, 198] Several of thes e proteins function as e n zymes, which facilitate HR repair For example, the MRN complex  has nuc lease activities and BACH1  and BLM  are DNA helicases The interaction of BRCA1 with PALB2 is of particular interest because the latter is also a b reast cancer predisposition gene [164, 201 203] Furthermore, biochemical st u dies on the BRCA1/PALB2 complex demonstrated that PALB2 not only binds to the coiled coil domain of BRCA1 through its own coiled coil doma in  but also int e racts with BRCA2 via the WD40 domain  Thus, PALB2 interacts with both BRCA1 and BRCA2 and acts as a bridge between them [164, 205]
47 Lastly, the participation of BRCA1 in the HR is demonstrated by the relation b e tween BRCA1 and Rad51 recombinase. According to the current understanding there is a hierarchy in the recruitment of the aforementioned proteins to the DNA damage sites. BRCA1 localization to the DSBs does not depend on PALB2, BRCA2, or Rad51 reco m binase. PALB2 is recruited to the sites of damage at least in part via its interaction with BRCA1  Furthermore, PALB2 interacts with BRCA2, the protein that loads the Rad51 reco mbinase on chromatin. T he role of BRCA2 in HR is a dir ect control of Rad51 nucleoprotein formation  while the role of BRCA1 seems to be more complex. BRCA1 functions earlier in HR and DNA damage checkpoint signaling by interacting with PALB2, BACH1, CtIP, and MRN complex In addition to their role in HR, BRCA1, BRCA2, and PALB2 participate in the i n tra S phase checkpoint. Cells defective in the expression of these proteins do not have intact intra S phase checkpoint [152, 205, 207] Therefore, the intra S phase checkpoint is another possible process except for HR, in which the protein complex that consists of BRCA1, BRCA2, and PALB2 can potential ly be involved. It is equally possible that BRCA1 participates in the intra S phase checkpoint signaling independently of BRCA2 PALB2 complex. At this stage, the mechanism by which BRCA1, BRCA2, and PALB2 carry out their tumor suppression activities is n ot clear. One possibility is that BRCA1 is localizing PALB2, BRCA2, and Rad51 at the DSBs, and consequently facilitating HR [121, 137] Another possibility is that BRCA1, PALB2, and BRCA2 alone or in combination a re r e sponsible for the intact intra S phase checkpoint signaling. Finally, it may happen that
48 all of the above mentioned possibilities are working together. However, it is important to point out that the coiled coil domain of BRCA1 seems to be necessary bo th for its role in HR  and for the intra S phase checkpoint function (Velkova a nd Monteiro unpu b lished results ). In addition to PALB2, another import ant interacting partner of BRCA1, involved in the control of HR, is CtIP. In mammalian cells, CtIP is phosphorylated by CDKs and it exists in a complex with BRCA1 in a cell cycle (during G2 phase) dependent manner [ 208] Studies performed on the avian B cell line DT40 demonstrated that CtIP partic i pates in micro homology directed end joining (during G1 phase) and in HR (during S and G2 phases)  Importantly, the function of CtIP exclusively in HR is dependent on its interaction with BRCA1 [178, 208, 209] Moreover, BRCA1 promotes CtIP ubiquitin a tion and its localization to the sites of DSBs  Both CtIP and BRCA1 control RPA foci formation after DNA damage, thus co n trolling ATR activation [198, 209, 211] In addition, BRCA1 co localizes with the MRN complex at the IR induced foci [120, 212] and inhibits its exo nuclease activity  while CtIP stimulates the nuc lease activity of the MRN complex  Based on these d ata, it has been proposed that BRCA1/CtIP/MRN complex participates and controls the initial processing and/or resection of DSBs  Furthermore, BRCA1 might participate in the end resection by its relation to 53BP1. Only recently, the details about the ir role in the DDR became clear. Two recent papers explored the effect of 53bp1 deletion in Brca1 deficient mouse cel ls. In the first report, the deletion of 53bp1 rescues several phenotypes associated with Brca1 deficiency
49 such as the proliferation defect, hypersensitivity to DNA cross linking agents, spontan e ous formation of DSB, and activation of the DDR  In the second report, 53bp1 d e letion rescues the HR defect of Brca1 mouse cells by a mechanism dependent on ATM controlled end resection  Thus, it has been hypothesize d that BRCA1 and 53BP1 might regulate the choice between HR and NHEJ  at least partially by their role in end resection. The manner in which BRCA1 in complex with CtIP, MRN, 53BP1, or other proteins particip ates and controls end resection and DNA damage si g naling rem ains obscure and is a subject for active research. Taken together, these data suggest that BRCA1 participates in HR by controlling protein localization and different activities during the end rese ction step. Despite the ev i dent contribution of the studies cited above they should be interpreted wi th caution. The experiments described above have been carried out in avian B cell line DT40 (in the case of CtIP) and in mouse embryonic fibroblasts (in t he case of 53BP1). DT40 cell line has a short cell cycle taken up mostly by S phase and high levels of HR  In addition, the survival of mouse embryonic stem cells is dependent on HR. However, the relative co n tribution of HR and NHEJ in repair and cellular responses to damage varies between sp e cies and in different cells  Thus, the physiol ogical importance of the effects of BRCA1 deficiency on breast and ovarian epithelial cells remains to be tested.
50 DNA Repair by NHEJ NHEJ is considered to be the major DNA repair pathway responsible for fixing IR induced DSB in mammalian cells  It is operational throughou t all cell cycle stages  and dependent on the Ku (Ku70/86 ) heterodimer, DNA PKcs, XRCC4, DNA Ligase IV, Artemis, and XLF  (Figure 3) It is generally accepted that NHEJ has several stages: detection of DSB, end processing, and ligation  In the process of detection of DSBs, the Ku heterodimer is the one recognizing the DSBs Upon DNA damage, Ku86 interacts with a conserved m o tif on DNA PKcs C terminus, which leads to the recruitment of DNA PKcs to DSBs and its activation  Over 16 phosphorylation sites have been described on DNA PKcs, although their role is still poorly understood. Nevertheless, D NA PKcs phosphorylation status is believed to influence its activity  The unphosphorylated form of DNA PKcs interacts with Ku86  Further, DNA PKcs autophosphorylation is required for NHEJ progression and subsequently for end processing  It has also been shown that Ku86 interacts with BRCA1 N terminus, and this complex formation is necessary for the rapid recruitment of BRCA1 at DSB sites  However, the downstream events in the DDR signaling in which BRCA1 participates are not clear. Fur thermore, the reports for the role of BRCA1 in NHEJ have been conflicting (Table 4) ranging from no significant difference (slight elevation) to compromised NHEJ. However, the cited reports used different cell lines (mouse versus human, e m bryonic stem cel ls versus breast epithelial cells), which express various BRCA1
51 Moreover, to measure the role of BRCA1 in NHEJ, different assays were used: from gene targeting and single chro mosomal DSB at the specific position to plasmid based in vivo and in v i tro assays (Table 4). In addition, plasmid based assays account for the total end joining activity and do not discriminate between classical and alternative end joining. Thus, based on the available data, the role of BRCA1 in NHEJ regulation might be cell and DNA break type specific. Table 4. Comparison of different assays for BRCA1 repair function. Cell type Assay HR NHEJ Refs Mouse embryonic stem cells line 236.44 Gene targeting De creased Proficient, Elevated  Mouse embryonic stem cells Gene targeting Decreased Increased  Mouse embryonic stem cell line 236.44 I Sce I induced chrom o somal break Decreased Proficient, Elevated  Mouse embryonic stem cells Cell free assay total end joining Decreased  HCC1937 Plasmid base in vivo end joining Decreased precise end joining  HCC1937 Plasmid base in vivo end joining Decreased precise end joining 
52 Hypothes e s for the Tissue Specific BRCA1 F unctions Several hypotheses have been proposed to address the tissue specific functions of BRCA1. First, Scully and Livingston  hypothesized that some estrogen metabolites could potentially form DNA adducts. Specifically, breast and ovarian epithelial cells are estrogen responsive. Thus, BRCA1 may play an essential role in the repair of estrogen induced DNA damage that cannot be compensated by other proteins. S econd, Elledge and Amon  proposed a hypothesis, according to which the loss of BRCA1 as an essential gene has two different end results. In various tissues except for the breast and ovary, the loss of BRCA1 leads to cell death or proliferation defects. Nevertheless, in the breast and ovary BRCA1 negative cells receive the necessary survival signals (horm ones, growth factors etc .) and continue to divide. This growth adva n tage increases the possibility of the secondary mutations to be accumulated over time, which in some cases will lead to the development of breast and/or ovarian cancer. In addition, Foul kes extended that BRCA1 functions as a major regulator for breast stem cells differentiation  In this role, BRCA1 regulates differentiation of the breast epithelium. Failure in this regul a tory mechanism results in loss of control over cell growth and potential block in the proper differentiation of the breast epithelial cells. This hypothesis explains some of the phenotype s of BRCA1 associated breast cancer (see page s 7 8 ). Finally, Monteiro proposed an alternative mechanism to explain the tissue speci f ic tumor suppressor r ole of BRCA1  According to this hypothesis, BRCA1
53 locus is the subject to increased loss of heterozygosity rates only in the breast and ovarian ep i thelial cells but not in the cells from the other tissues. Currently, none of these models has been experimentally tested and verified. Summary and R ationale Women who are carriers of deleterious mutations in BRCA1 are at a greater risk to develop brea st and ovarian cancer than women from the general population. The 17 years after the cloning of BRCA1 have been fruitful for breast cancer research. The result s from such research benefit patients not only when they make informed clinical decisions, but al so when they undergo therapy for their BRCA1 related breast cancer. However, the e x perience from the bench and from the clinical practice shows that much more remains to be learned about BRCA1 and the mechanism through which it participates in one of the m ost universal processes in life maintenance of the genome stability. Considering that there are over 1500 alleles of BRCA1 in the human population one of the most challenging tasks for genetic counseling remains to distinguish which are benign and whi ch are cancer predisposing. Previous research has indicated that the lik e lihood of a BRCA1 variant being deleterious is higher when the variant is located in structurally and functiona lly defined protein domain s such as the RING and BRCT d o mains Consequen tly the identification of other functional domains, besides the well studied ones, is critical for the understanding of BRCA1 function and for classifying variants as cancer predisposing or benign To approach this problem we formulate our
54 ce n tral hypoth esis : poorly characterized, conserved domains in the central region of BRCA1 (Motif2, Motif 6 and coiled coil domain) directly participate in the tumor suppression functions of BRCA1. This dissertation aims at test ing our hypothesis and determin ing how s pecific d o mains and motifs of BRCA1 act to promote tumor suppression. In particular, we set out to determine the functional significance of the two poorly characterized domains of BRCA1. This is a step further in our long term goal to understand the mechan isms by which BRCA1 is required for maintaining genomic stability and to use this information to develop a system of assays for testing the functional role of any mutation in BRCA1. Importantly, our research has much broader implications because the gene products i n volved in breast cancer seem to cluster around DNA damage response pathways. Thus, the understanding of BRCA1 role may have an impact on other forms of breast cancer not attributable to germline mutations in BRCA1. Moreover, b oth radiation the rapy and most of the drugs used for cancer treatment rely on introducing DNA damage in the cells. BRCA1 is the main participant in the cellular response to DNA damage, which makes it an important factor in
55 MATERIALS AND METHODS Constructs and C loning GST fusion fragments of BRCA1 in the mammalian expression vector pEBG BF 1 6 were a gift from Toru Ouchi. BRCA1 fragments BF1A (aa 1 70), BF1B (aa 71 140), BF1C (aa 1 101), BF1D (aa 141 240), BF1D1 (aa 1 60 190), BF1D2 (aa 190 210), BF1D3 (aa 160 210), BF1E (aa 241 324), and BF1F (aa 1 302) were obtained by PCR using pEBG BF1 as template. The PCR products were digested, cloned into pEBG ve c tor, and sequenced. Construct BF1D Y179C was obtained by site direc ted mutagenesis using BF1D as template for the PCR reaction. FLAG FLNA Bf was obtained by cloning a PCR fragment of Filamin A (FLNA, aa 2477 2647) in frame to FLAG in pCMV2 FLAG vector. For the yeast two hybrid interactions assays BRCA1 Motif 6 fragment ( aa 845 869) was obtained by PCR and cloned in frame with the GAL4 DNA binding domain i n to pGBKT7 vector. Similarly, FLNA repeat 23/Hinge/repeat 24 (aa 2477 2647), FLNA repeat 23 (aa 2477 2516), FLNA Hinge (aa 2517 2549), FLNA repeat 23/Hinge (aa 2477 2549) FLNA repeat 24 (aa 2550 2647), FLNA repeat 24 (aa 2517 2647) were
56 o b tained by PCR and cloned in frame with GAL4 activation domain into pGAD 424 vector. The constructs containing BRCA1 Motif 6 mutants: S864L, F861C, Q855P, R866C, and R866H were obtained b y site directed mutagenesis using as a template for the PCR reaction pGBKT7 M otif6 (aa 845 869) The products were digested, cloned in frame with GAL4 DNA binding domain into pGBKT7 vector, verified by sequencing, and used for the transformation of yeast s train AH109. Yeast Two Hybrid A ssays For deletion analysis the yeast two hybrid was performed using the MA T CHMAKER Two Hybrid System 3 (Clontech laboratories). In brief, pGBKT7 Motif 6 and pGAD 424, containing FLNA deletion mutants were co transformed in AH 109 yeast strain. Transformations were plated on SD Leu Trp selective media to access the eff i ciency of the transformation and SD Leu Trp Ade His to map the minimal domain of FLNA necessary for binding to BRCA1. For the liquid culture growth assay t he AH109 transformants were inoculated in liquid media lacking either leucine and triptophane ( leu, trp, double se lection) or lacking leucine, tryptophan histidine, and adenine ( leu trp his ade, quadruple selection). The vectors used for the exp ression confer growth in the abs ence of leucine (pGAD424) or tryptophan (pGBKT7). Samples were taken every two hours and quantified by spectr o photometer at a wavelength of 600nm. The growth on double selection was used
57 to e n sure that the constructs tested are not toxic for the yeast. If any of the Motif 6 constructs interact with FLNA construct activation of the expression of histidine and adenine will follow and the yeast will grow in media lacking histidine and adenine. Cell Lines and T ransfections The FLNA deficient M2 melanoma cell line and its isogenic cell line, A7, recon s tituted with full length FLNA cDNA [227 ] (gift from T. Stossel) was grown in MEM (Sigma) with 8% newborn calf serum (Sigma) and 2% fetal bovine serum (FBS; SAFC Biosciences, Lenexa, KS). A7 cells were grown in the presence of 0.2 mg/ml G418 (Fisher). HeLa (ATCC, Manassas, VA) was grown in DMEM with 5% FBS 293FT (InVitrogen) cells were grown in DMEM media (Sigma) with 10% FBS. Tissue culture media was supplemented with penicillin and streptomycin. Transfections were performed using Fugene 6 (Roche) according to th Antibodies The following antibodies, peptides, and beads were used: BRCA1 mouse m o noclonal antibody MS110 (Ab 1; Calbiochem; San Diego, CA) and SG11 (gift from D. Livingston); Filamin A mouse mo noclonal antibody PM6/317 (Chemicon Internatio n al); FLAG M2 mouse monoclonal antibody (Sigma); 3xFLAG peptide
58 (Sigma); GST goat polyclonal antibody (Pharmacia Biotech); GT sepharose 4B beads (GE Healt h care); Ku86 monoclonal antibody B 1 and Rad51 r abbit polyclonal antibody H 92 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); TP53BP1 mouse monoclonal ant i body and phosphoserine 343 NBS1 mouse monoclonal antibody (Upstate Biotechno l ogy); p34 RPA mouse monoclonal antibody Ab 1 and DNA PKcs mou se monoclo n al antibodies Ab 2 (Neomarkers, Freemont, CA); phosphoserine 2056 DNA PKcs ra b bit polyclonal antibody (Abcam, Cambridge, MA); phosphoserine 2609 DNA PKcs rabbit polyclonal antibody (Novus Biologicals); MDC1 (SIGMA); H2AX rabbit p o lyclona l; H2AX; phosphoserine 1981 ATM; ATM; ATR; phosphothreonine 68 CHK2; phosphoserine 317 CHK1 (Cell Signaling); actin (Sigma). Conjugates for immunofluorescence were Alexa fluor 488 or 555 Molecular Probes. Immunoprecipitation, Pull Downs, We stern Blot Analysis, and D ensitometry Whole cell extracts were prepared by lyzing cells in a mild RIPA buffer (120mM NaCl, 50 mM Tris pH 7.4, 1% NP40, 1mM EDTA, protease inhibitors, 4 mM PMSF) lacking harsher SDS, sodium deoxycholate, and Triton X 100 det ergents The same bu f fer was used for immunoprecipitation. For high stringency immunoprecipitations the R I PA buffer was supplemented with 0.5 % SDS. Antibodies (1 g) were pre incubated with protein A/G agarose beads (Santa Cruz Biotechnology, Inc.), washed twice in RIPA bu f fer and incubated with the cell extracts overnight at 4C. After incubation, the slurry
59 were pelleted by centrifugation (2,000 rpm) and washed twice by removing the supernatant. Sample buffer was added to the beads and boiled for 10 min. For GST pull downs, cell extracts were incubated with GT beads, washed in RIPA buffer, and boiled. Samples for western blot analysis were separated by SDS PAGE and gels were electroblotted on a wet apparatus to a PVDF membrane. The PVDF membrane was blocke d with 5% milk in TBS buffer containing 0.1% Tween (TBS Tween) for 1h. The membrane was washed three times in TBS Tween and the antibody was added in 0.5% milk in TBS Tween. The membrane was washed three times in TBS Tween and inc u bated with the appropriat e conjugate. After final washes Blots were incubated with ECL (Millipore, Billerica, MA). Chromatin fractions were obtained by lyzing the cells with mild RIPA buffer and centrifuging at 14,000 rpm for 5 min. The pellet was then washed twice in mild RIPA a nd extracted with acid extraction buffer (0.5M HCl, 10% Glycerol, 100mM BME) and su b sequently neutralized using 40mM Tris pH 7.4 with protease inhibitors and NaOH. Wes t ern blot data was quantified by densitometry using AlphaEaseFC v 3.1.2. Each lane was no rmalized using the corresponding loading controls and then expressed as a fold change relative to the untreated FLNA + cells in each blot.
60 Immunofluorescence For BRCA1 analysis cells were fixed with 4% formaldehyde for 5 min followed by 5 min incubati on with 100% ethanol. Cells were permeabilized with 0.25% Triton X 100 in PBS for 10 min, washed with PBS, and then blocked for 30 min with 5% BSA in PBS at room temperature (RT). After blocking, BRCA1 monoclonal antibody (SG11; kind gift from David Living ston) was added to 1% BSA in PBS for 1h RT. Cells were washed and goat mouse Alexa Fluor 488 (Molecular Probes) was added for an add i tional 1h RT. For all other antibodies, cells were plated onto chamber slides and after 24h they were washed with cytosk eleton buffer (10mM HEPES/KOH pH 7.4, 300mM sucrose, 100mM NaCl, 3mM MgCl 2 ) and fixed with 4% formaldehyde for 30 min RT. For anal y sis of chromatin bound RPA cells were pre extracted for 2 min on ice with cytoskeleton buffer supplemented with 0.5% Triton X 100 before fixation  After fixation cells were permeabilized with 0.25% Triton X 100 in PBS for 5 min RT and then washed and blocked with 5% BSA in PBS for 30 min RT. Primary and secondary antibodies in 1% B SA in PBS were added for 1h each. Cells were washed and mounted with Prolong Gold medium (Molecular Probes). Images were taken on a Leica Confocal Microscope. For quantification of BRCA1 and Rad51 immunofluorescence foci approximately 100 cells were score d per each time point. Cells were scored as foci positive if they presented with more than 10 foci per cell (an e xample can be found in Figure 34 ). For H2AX and NBS P 343 at least 50 cells per
61 time point were counted for each condition. Cells were scored as foci positive if they pr e sented with more than 20 foci per cell. A threshold of 20 foci was chosen based on the number of foci found in unirradiated samples using the described antibodies. Determin a tion of foci number per cell was done using Definiens Developer XD 1.1 (Definiens AD, Germany). A rule set was developed to segment nuclei based on the DAPI stain and then segment foci within the nucleus based on an intensity threshold. Representative results from at least two independent experiments are sho wn instead of statistical data on a small number of measurements with variability as recently recommended  Comet A ssay Comet assays were performed in neutral conditions using a comet assay kit (Tr e vigen, were collected at the indicated time points, combined in low melting agarose (Trevigen, Ga i thersburg, MD), spread over the comet slide area and allowed to set. Then, slides were im mersed in lysis buffer for 30 min at 4C. Electrophoresis was run in TBE buffer for 20min at 1V/cm voltage. Image analysis was done with Comet Analysis System 2.3.3 software (Loats Associates Inc., Westminster, MD).
62 RESULTS Characterization of the I nteraction b etween FLNA and BRCA1 Identification of FLNA as a Binding P artner of BRCA1 Motif 6 in Y east Our laboratory has focused on the systematic analysis of domains and motifs in BRCA1 as a means to understand its biochemical functions  Here we analyzed a conserved region, called Motif 6, spanning amino acids 845 869 coded by large exon 11 (Figure 5 ) [94, 145] In order to identify interactors for the con served Motif 6 of BRCA1 spanning amino acid residues 845 869 (Figure 9 ) we performed a yeast two hybrid screening against a human mammary gland cDNA library. Two overlapping clones coding for h u man Filamin A (FLNA; OMIM # 300017), spanning amino acid resi dues 2443 2647 and 2477 2647 (Figure 9 ), were identified. This region includes repeat 23, the hinge region, and repeat 24 in the C terminus FLNA (Figure 9 )  We mapped the minimal region of FLNA, which interacts with BRCA1 Motif 6 b y testing binding of a series of FLNA del etion mutants (Figure 9 ). Only the fragment aa 2477 2647 was able to bind to BRCA1 Motif 6 (Figure 9). Interestingly, FLNA has been shown to interact with BRCA2
63 and to participate in the DDR [231 233] Cells lacking FLNA exhibit prolonged checkpoint a c tivation leading to the accumulation of cells in G2/M after ionizing radiation  Interaction b etween FLNA and BRCA1 in Mammalian C ells Next, we tested whether endogenous FLNA interacted with endogenous BRCA1 in mammalian cells. Immunoprecipitation using a specific monoclonal antibody against BRCA1 pulled down FLNA in HeLa and HCT116 cells (Figure 10 ). In addition, imm u noprecipitat ion using an antibody against FLNA was able to pull down BRCA1 (Figure 10 ). Thus, BRCA1 and FLNA interact in vivo and the interaction is mediated by the C terminus of FLNA. Because FLNA and BRCA1 have been demonstrated to be primarily cytoplasmic and nuc lear respectively, we biochemically fractionated HCT116 cells to determine in which subcellular compartment t he interaction occurs (Figure 11 ). We found that FLNA is expressed in the nucleus and cytoplasm and BRCA1 can be co immunoprecipitated by FLNA i n the nuclear fraction (Figure 11 ). We also determined that the interaction is d i rect as bacterially expressed GST tagged BRCA1 (aa 141 302) can pull down bacterially expressed His t agged FLNA C terminus (Figure 12 ).
64 Figure 9. BRCA1 Motif 6 binds to Filamin A (aa 2477 2674) in yeast. Top panel: Diagram showing a Filamin A dimer. Red box, Actin binding domain; blue box ind i cates the minimal region that interacts with Motif 6 of BRCA1. Note that this region contains the dimerization domain for two Filamin A subunits. Bottom panel: Yeast two hybrid experiments testing Motif 6 interaction with Filamin A fragments, as well as positive (La rge T and p53) and negative (FLNA and empty vector) controls. Smal l er regions of FLNA such as repeat 23, hinge region, repeat 23 and hinge, repeat 24, and repeat 24 plus hinge failed to interact indicating that both repeats and the hinge region are necessa ry for a stable interaction.
65 Figure 10. BRCA1 and Filamin A interact in mammalian cells. Upper panels, co immunoprecipitation of endogenous BRCA1 with FLNA in HeLa and HCT116 cells showing interaction of endogenous BRCA1 and FLNA. Lower panels, reverse rea c tion showing co immunoprecipitation of endogenous FLNA with BRCA1.
66 Figure 11. FLNA interacts with BRCA1 in the nucleus. HCT116 cells were fra c t ionated into nuclear and cytoplasmic fractions using PARP and Talin as controls, respectively. Top panel shows that BRCA1 can be immunoprecipitated using FLNA antibody in the nuclear fraction.
67 Figure 12. FLNA directly binds to BRCA1 in vitro. GST and GST BRCA1 (aa 141 302) were used in pull down in vitro assays. GST BRCA1 but not GST can pull down bacterially expressed His tagged FLNA C terminal region.
68 BRCA1 Binding to FLNA is Mediated P rimarily by BRCA1 Motif 2 not Motif 6 We searched the Breast C ancer I nformation Core Database ( BIC, http://res earch.nhgri.nih.gov/projects/bic ) and identified five missense variants in BRCA1 Motif 6 region: S864L, F861C, Q855P, R866C, and R866H. All five mutants were class i fied as VUS s Next, we tested the extent to which the indicated variants interact with FLNA We checked the ability of the yeast strain AH109 co transformed with constructs coding for FLNA and Motif 6 to grow on liquid media under stringent selection If any of the Motif 6 constructs interact ed with FLNA construct the activation of histidine a nd adenine expression would follow and the yeast would grow in media lacking histidine and adenine. In this experiment a strong interaction should have similar profile as the positive control. However, e ven the wild type Motif 6 did not display such a p rofile, indicating that t he inter action s between FLNA and all Motif 6 constructs tested w ere rel a tively weak (Figures 13 and 14 ). This observation raises questions about the significance of the results obtained with the Motif 6 mutants. Thus, we hypothesi zed that other regions in BRCA1 besides Motif 6, might contribute to BRCA1 binding to FLNA We co expressed in frame fusions of GST to deletion fragments of BRCA1 and a FLAG tagged FLNA fra g ment (aa 247 7 2647) in 293FT cells (Figure 15 ) to assess each reg contribution to binding. We immunoprecipitated FLAG FLNA using FLAG agarose beads and the eluate with FLAG peptide was separated by SDS PAGE. Western blot against endoge n ous FLNA confirmed that FLAG FLNA was properly folded because FLAG
69 Figure 13. Growth curve in medium lacking leucine and tryptophan S.cerevisiae strain AH109 (Clontech) was transformed simultaneously with pGBKT7/ Motif 6 m u tants (aa 845 869) and pGAD424/ FLNA (aa 2477 2 647) constructs. Growth curves i n medium lacking leucine and try pt ophan ( leu, trp, double selection) are shown. OD 600 of the cultures (y axis) is plotted against time (x axis). Large T Antigen (aa 86 708) and p53 (aa 72 309) were used as a positive contro l and pGBKT7/Motif6+ pGAD424 and pGAD424/FLNA+pGBKT7 as negative controls. The growth on double selection was used as a control to e n sure that the constructs tested are not toxic for the yeast.
70 Figure 14. Interaction between pGBKT7 Motif 6 mutants (aa 845 869) and pGAD424 FLNA (aa 2477 2647) constructs. S.cerevisiae strain AH109 (Clontech), transformed with the indicated constructs, was grow n in medium lacking leucine, try pto phan histidine, and adenine ( leu trp his ade, quadruple selection). OD 600 of the cu l tures (y axis) is plotted against time (x axis ). Large T Antigen (aa 86 708) and p53 (aa 72 309) were used as a positive control and pGBKT7/Motif6+pGAD424 and pGAD424/FLNA+pGBKT7 as negative controls. Note that none of the yeast, transformed with the M6 /FLNA constructs grew within the first 24 hours, indicating that the interaction between BRCA1 Motif 6 and FLNA is weak.
71 Figure 15. Expression of BRCA1 fragments in 293FT cell line. Left panel, diagram of deletion constructs used to map the BRCA1 interaction site to FLNA. RING, RING finger domain; NLS, nuclear localization signals; BRCT, BRCA1 C terminal domains. Right panels, co expression of GST fragments of BRCA1 and FLAG FLNA ( aa 2477 2647) in 293FT cells. Lower molecular weight band obtained in the empty vector (V) transfection corresponds to GST.
72 Figure 16. FLNA interacts with multiple sites in BRCA1. Co immunoprecipitation of BRCA1 fragments (WB GST), endogenous BRCA1 (WB BRCA1) with FLAG FLNA ( aa 2477 2647). Note that endogenous FLNA is also immunoprecipitated by FLAG FLNA ( aa 2477 2647) confirming it is in the native conformation (WB FLNA). Strong reactivity shown for GST BF1 is due to recognition to the GST BRCA1 fragment that contains the epitope for the antibody.
73 Figure 17. GST pull down experiments show that GST BRCA1 fragments 1, 3, and 4 can precipitate endogenous FLNA. Co immunoprecipitation of BRCA1 fra g ments (WB GST) and endogenous FLNA (WB FLNA).
74 Figure 18. FLNA binds to aa 141 240 in BRCA1. Upper panel, di agram of deletion constructs used to map the interaction site to FLNA. NES, nuclear export sequence (black boxes). Lower panels, FLAG FLNA ( aa 2477 2647) interacts strongly with GST BRCA1 fragments BF1 (aa 1 324), BF1D (aa 141 240), and BF1F (aa 1 302).
75 F igure 19. FLNA binds to aa 160 190 in BRCA1. Upper panel, diagram of deletion constructs of fragment aa 141 240 used to map the interaction site to FLNA. The loc a tion of the missense variant Y179 is indicated. Middle left panel, FLAG FLNA ( aa 2477 2647) co immunoprecipitates with GST BRCA1 fragments BF1D1 (aa 160 190) and BF1D3 (aa 160 210). V, GST; D1, GST BRCA1 fragment BF1D1 (aa 160 190); D2, GST BRCA1 fragment BF1D2 (aa 190 210); D3, GST BRCA1 fragments BF1D3 (aa 160 210). Bottom panel, control for expr ession levels. Middle right panel, GST BRCA1 fragments BF1D1 (aa 160 190) and BF1D3 (aa 160 210) can pull down end o genous FLNA.
76 FLNA co n tains the dimerization domain for two FLNA subunits (Figure 9). In addition, western blot against BRCA1 showed that FLAG FL NA interacts with endogenous BRCA1 (Fi g ure 16, left panel). Moreover, western blot against GST revealed the interaction of FLNA with different fragments of BRCA1 under low stringency (Figure 16, left panel). Interaction with Fragments 1 (aa 1 324), 3 (aa 502 802), and 4 (aa 758 1064) was detected even under high stringency conditio ns (Figure. 16 right panel). R e verse pull downs of endogenous FLNA using GT beads confirmed that the interaction is mediated by BRCA1 F ragments 1, 3, and 4 (Figure 17 ). In both experiments, BRCA1 Fragment 1 showed the s trongest interaction (Figures 16 and 17 ). Fragment 1 (aa 1 324) includes the RING finger (aa 1 101)  and nuclear e x port signals (aa 22 30 and aa 81 99) [144, 234] To determine whether the interaction was mediated by these motifs, we used deletion mutants of BRCA1 F ragment 1 (Figure 18 ). Initially, we identified BRCA1 residues 141 240 as the interacting region to FLNA (aa 2477 264 7) (Figure 18 ). Further mapping identified residues 160 190 as the minimal r e gion required for binding (Figure 19 ). This region, called Motif 2, had been previously identified as a conserved motif in BRCA1 orthologs [94, 145]
77 BRCA1 Variant Y179C, Found in Breast and Ovarian Cancer Patients Disrup ts the Binding b etween BRCA1 and FLNA To assess whether BRCA1 and FLNA interaction might contribute to breast ca n cer we searched the Breast Cancer Information Core database ( http://research.nhgri.nih.gov/bic/ ) for variants in this region. Variant Y179C is a frequent missense change recorded in the database (BIC Database). The i ntroduction of BRCA1 Y179C mutant significantly reduced BRCA1 interaction to FLAG FLNA aa 2477 2647 a nd to endogenous FLNA (Figure 20 upper and middle panels ) further demonstrating the specificity of the interaction. Because other regions in BRCA1 except for Motif 2 also contributed to the binding we investigated whether the Y179C mutation would disrupt binding to FLNA in the context of full length BRCA1. The i ntroduction of Y179C mut a tion significantly reduced the interaction in the full length context as comp ared to wild type BRCA1 (F ig. 19 bottom panel). In summary, these experiments demonstrate that Motif 2 primarily me diates the interaction with FLNA. Taken together these data raised the possibility that lack of FLNA might impair BRCA1 foci formation after DNA da m age. Thus, the fo llowing experiments were directed at assessing the role of FLNA/BRCA1 interaction in the DNA damage response.
78 Figure 20. Introduction of BRCA1 Y179C mutation significantly reduces BRCA1 interaction to FLAG FLNA aa 2477 2647 and to endoge nous FLNA. W, wild type GST BRCA1 fragment BF1D (aa 141 240); Y, GST BRCA1 fragment BF1D with Y179C mutation. Bottom panels Introduction of BRCA1 Y179C mutation into a full length BRCA1 context significantly reduces interaction to endogenous FLNA. W, wild type full length BRCA1; Y, full length BRCA1 carrying a Y179C mutation.
79 Analysis of the DNA Damage R e sponse Signaling in FLNA Positive and Negative Cell L ines FLNA Deficiency Does Not Cause a Defect in Sensing DNA D amage To further characterize the functional significance of FLNA/BRCA1 interaction we obtained the M2 melanoma cell line which lacks FLNA and its counterpart A7 which was obtained by reconstituting M2 cells with full length FLN A cDNA  First, we assessed the kinetics of DSB repair after IR. We irradiated or mock treated the FLNA and FLNA + cell lines and collected cells at several time points after IR. We monitored the presence of DSB with an antibody against histone H2AX phosphorylated at Serine 139 ( H2AX), a marker for DSBs  Whereas the FLNA + cell line efficiently repaired DSBs and by 8h after IR there was no detectable H2AX (Figure 21 ), FLNA cells had a sustained high level of H2AX for up to 32h after IR. We confirmed this observati on using Comet assays (Figure 22 ).
80 Figure 21. FLNA null cells are deficient in DNA repair. FLNA + (A7) and FLNA (M2) cells were irradiated with 8 Gy or mock treated (U) and harvested at the ind i cated time points. Wh ile FLNA + cells repair most of the DSBs (as measured by H2AX) by 8h, FLNA cells show significant unrepaired DSBs even after 32h post IR ( Top panel) Bottom panel shows total levels of H2AX as a loading control.
81 Figure 22. Neutral comet confirms that FL NA null cells are deficient in repair. FLNA + (A7) and FLNA (M2) cells were irradiated with 8 Gy or mock treated (NO IR) and harvested at the indicated time points and comet assays were performed u n der neutral conditions. A two performed and p values are shown for statistically significant differences.
82 Further we assessed whether cells lacking FLNA had a compromised DNA da m age signaling. Thus, we tested whether ATM and ATR were properly activated upon DNA damage. The p hosphorylation of ATM S1981 was not compromised i n FLNA cells (Figure 23 Top panels). Likewise, the phosphorylation of CHK2 T68 and CHK1 S317, markers of ATM and ATR activation respectively, did not show a defect (Figure 24 ). Surprisingly we consistently observed higher levels of phosphorylation of AT M, CHK2, and CHK1 in cells lacking FLNA (Figures 23, 24 ) which indicat ed the upregulation of ATM and ATR signaling. These results confirmed the previous data by Meng et al  showing the sustained activation of CHK2 and CHK1 in FLNA deficient cells following damage. The Defect in FLNA Deficient Cells I s n ot Restricted to Ionizing R adiation To determine whether the repair defect we observed was restricted to double strand breaks caused by IR we tested a panel of agents that cause DNA damage by di f ferent mechanisms. First, we tested Camptothecin (CPT) a specific topoisomerase I inh i bitor, by incubating cells with the drug for 1h and then removing it. The c ells were then collected at several time points after th e drug removal. Similar to what was found with IR we noted that at early time points FLNA cells displayed increased and sustai ned act i vation of ATM (Figure 25 ). CHK2 also retained high levels of phosphorylation up to 6h after the drug removal in FLNA ce lls (Figure 25 ). Next, we investigated the phosphor y lation status of DNA PKcs at residues S2056 and S2609, which represent the
83 two main SQ/TQ clusters of phosphorylation  Interestingly, while levels of phosphorylation in S2069 are similar in both cell lines, phosphorylation lev els of S2056 were markedly diminished in FLNA cells (Figure 25 ). In addition, we saw the increased and sustained phosphorylation of CHK1, an indirect measure of ATR activation, and persistent pho s phorylation of NBS1 even at 6h time point after the drug re moval in cells lacking FLNA (Figure 26 ). The p hosphorylation of RPA which can be seen as a slower migrating ban d above the main band (Figure 27 ) was also increased and sustained up to 24h in FLNA cells. Further we tested the effect of Hydroxyurea (HU ), a replication inhibitor that targets ribonucleotide reductase, by incubating cells with the drug for 24h and then remo v ing it. The c ells were then collected at several time points after the drug removal. In ge n eral, we verified that FLNA cells treated with HU displayed the increased and sustained phosphorylation of CHK2, CHK1, NBS1, and RPA (Figures 28 29 ). These data are co n sistent with the abnormal activation of the DNA damage checkpoint after CPT and HU treatment s
84 Figure 23. FLNA null cells show no impairment in activating the DNA damage response. Top two panels, ATM activation as measured by phosphorylation of S1981 is not compromised in F LNA cells. Blot for total ATM is used as a loading control. Note significantly higher levels of pS1981 ATM in FLNA cells. Middle three panels, no significant difference was observed in levels of DNA PKcs S2056 or S2609 phosph o rylation but recruitment of DNA PKcs to chromatin is defective in FLNA cells. Bottom two panels, ATR presence in chromatin (CHR) is shown. Blot for ATR levels in whole cell lysates is used a loading control.
85 Figure 24. CHK2, CHK1, and NBS1 activation as measured by pT68 CHK2, pS317 CHK1, and pS343 NBS1, respectively is not compromised in FLNA cells. Note consistently higher levels of pT68 CHK2, pS317 CHK1, and pS343 NBS1 in FLNA cells. actin is used a loading control.
86 Figure 25. Effects of the CPT treatment on the DNA damaging signaling in FLNA deficient and proficient cell line ATM, CHK2 and DNA PKcs activation. Note increased phosphorylation levels of S1981 in ATM in earlier time points found in FLNA cells after Camptothecin (CPT) treatment (first panel). FLNA cells also di splayed retention of phosphorylation of T68 in CHK2 even at 6h after drug removal (second panel). Levels of Ku86 were used as loading control (third panel panel). FLNA deficient cells also display diminished levels of DNA PKcs S2056 phosphor y lation (fourth panel) but not of S2609 (fifth panel). Total levels of DNA PKcs were used as loading control (sixth panel).
87 Figure 26. Effects of CPT treatment on the DNA damage response signaling in FLNA deficient and proficient cell line CHK1 and NBS1 activation. Leve ls of phosphoserine 317 in CHK1 progressively increase in FLNA remaining elevated even at 6h after drug removal (Top panel). Note also the retention of high levels of phosphorylation at S343 in NBS1 (middle panel). Levels of actin were used as loading c ontrol (bottom panel).
88 Figure 27. Effects of CPT treatment on the DNA damage response signaling in FLNA deficient and proficient cell line RPA phosphorylation. Deficiency in FLNA lead to increased and sustained phosphorylation of chromatin bound RPA as me asured by the slower migrating band of RPA (top panel). Levels of Histone H2AX are used as loading controls (bottom panel).
89 Figure 28. Effects of HU treatment on the DNA damage response signaling in FLNA deficient and proficient cell line CHK1, CHK2 and N BS1 activation. Note increased phosphorylation levels of T68 in CHK2 found in FLNA cells after treatment with Hydroxyurea (first panel). FLNA cells also displayed retention of phosphorylation of S317 in CHK1 even at 6h after drug removal (second panel). Levels of Ku86 were used as loading control (third panel). FLNA also displayed the retention of high levels of phosphorylation at S343 in NBS1 in late time points (fourth panel).
90 Figure 29. Effects of HU treatment on the DNA damage response signaling in FLNA deficient and proficient cell line RPA phosphorylation. Deficiency in FLNA also lead to increased and sustained phosphorylation of chromatin bound RPA after Hydroxyurea treatment (top panel). Levels of Histone H2AX were used as loading controls (botto m panel).
91 FLNA Deficiency Impairs BRCA1 and Rad51 Foci F ormation To determine whether the deficiency in repair was due to the defective recrui t ment of factors required for the DDR we performed immunofluorescence analysis in non irradiated or irradiated cells at 1 h and 24h time points after IR. The a ccumulation of H2AX and pS343 NBS1, early markers of DNA damage, was comparable in both cell lin es at 1h time point (Figure 30 ). In order to determine if there were small differences we quantifie d foci positive cells (Figure 30 lower panel). The r esults were comparable in both cell lines at 0 and 1 h after IR, but FLNA negative cells showed the inc reased nu m ber o f foci positive cells after 24h Likewise, the recruitment of mediator proteins MDC1 and 53BP1 was also comparable at 1h time point (Figure 31 ). Finally, the repair factor RPA did not show any difference between the cell lines at 1h time poi nt (Figure 32 ). Consistent with our western blot results (Figure s 21 and 24 ) where we detected abno r mally high levels of H2AX, pT68 CHK2, and pS317 CHK1 at 24h time point we d e tected persistent foci of H2AX, pS343 NBS1, and RPA at 24h time point after irradi a tion only in FLNA deficient cells (Figures 30, 31 and 32 ). Thus, the repair defect in FLNA deficient cells was not due to a failure in to initiat ing the DNA damage response. Next, we investigated the ability of BRCA1 and Rad51 to form IR induced fo ci. The criteria for scoring cells as foci positive and foci negative are shown on Figure 34 The detailed analysis showed that FLNA deficient cells are unable to efficiently form BRCA1 IR induced foci as compared to FLNA proficient cells (Figure 31, botto m panel).
92 Figure 30. FLNA null cells show no impairment in recruiting DNA damage r e sponse factors to IR induced foc i H2AX and NBS1. Early markers of DNA damage H2AX (red) and phosphoserine 343 NBS1 (green) form foci irrespective of FLNA status. Note maintenance of foci after 24h only in FLNA cells. Lower p a nels show quantification of foci positive cells ( 20 foci).
93 Figure 31. Recruitment of DNA damage response mediator proteins 53BP1 (red, top panel) and MDC1 (green, middle panel) and BRCA1 to foci Recruit e ment of 53BP1 and MDC1 is comparable at early time points. BRCA1 foci form a tion was compromised in FLNA deficient cells (lower panel).
94 Figure 32. Recruitment of RPA and Rad51 to IR induced nuclear foci. Recruitment of repair factor p34 RPA (gree n, top panel) did not show any difference between the cell lines at the early time points. FLNA deficient cells displayed a delayed kinetics of Rad51 foci formation (bottom panel).
95 Although Rad51 displayed a comparable initial response at 3h time point after IR, it failed to mount a response comparable to FLNA proficient cells at 6h time point after IR. Rad51 p resented a delayed kinetics of foci formation with the peak at 15h time point in FLNA deficient cells (Figure 32 bottom panel). Taken together these data suggest that the compromised repair capacity in FLNA deficient cells may, at least partially, be m e ch anistically tied to the inefficient HR. The Lack of FLNA Leads to the A ccumulation of ssDNA a fter the DNA D amage During our analysis we noted that RPA foci in FLNA deficient cells were not only persistent 24h after the DNA damage but also significantly larger (Figure 32 ). To d e termine whether the RPA foci were associated with chromatin we pre extracted cells with Triton X100 before fixation. This method has successfully been used to detect only tightly bound to chromatin fraction of RPA  Interestingly, FLNA def icient cells accumulate large chromatin bound RPA foci whereas FLNA + cells present fewer and smaller chromatin bound RPA foci at 24h time point after IR (Figure 33 ). While most FLNA + cells have recovered from G2/M arrest and represent an asynchronous popu lation at 24h time point most FLNA cells remain arrested in G2/M at 24h time point after IR  Thus, these large tracts of ssDNA found in FLNA cells are unlikely to be due to the replication foci.
96 Figure 33. FLNA deficient cells present with large chromatin bound RPA fo ci at 24h after IR. Higher magnification of FLNA + and FLNA cells after 24h post IR. Left panel shows FLNA + cells stained for RPA (green). Middle panel shows FLNA cells stained for RPA (green). Note that nuclear foci are significantly larger than in FLNA + cells. Right panel shows a blow up of the inset (white square in middle pa n el) with staining for DAPI (blue), RPA (green), and H2AX (red).
97 Figure 34 Criteria for scoring foci positive cells Examples of foci positive cells for BRCA1 and Rad51.
98 Analysis of the Expression of BRCA1 Interacting Fragment of FLNA and FLNA Interacting Fragment of BRCA1 in FLNA Positi ve Cell L ine Expression of BRCA1 Interacting Fragment of FLNA Phenocopies the L oss of FLNA To gain more insight into the mechanism by which FLNA participates in DNA repair we transfected FLNA + and FLNA cell lines with flag tagged Filamin A aa 2477 2647 construct (BRCA1 interacting fragment). For the sake of simplicity we will refer to this FLNA B RCA1 interacting f ragment as FLNA Bf. At 24h post transfection we irr a diated cells with 8Gy IR and collected samples at different time points. The t ransfecti on of FLNA Bf did not lead to the checkpoint recovery in FLNA cells as measured by the phosp h orylation of CDC2 Y15 (Figure 35 ). Interestingly, transfection of the same fra g ment in FLNA + cells led to a similar phenotype as that found for FLNA cells as sho wn by phosphorylation of C DC2 Y15 and H2AX S139 (Figure 36 ). We also confirmed that expression of FLNA Bf acts in a dominant negative fashion in a stable tr ansfection co n text (Figure 37 ). We generated HCT116 cells stably expressing GFP FLNA Bf or GFP alone that were mock treated and irradiated. Cells expressing GFP FLNA Bf retained high levels of phosphorylated H2AX up to 32h after damage while cells expressing GFP alone showed levels returning to unirradiated leve ls at 8h after damage (Figure 37 ).
99 Figure 35. Expression of BRCA1 interacting fragment of FLNA in FLNA is u n able to reverse the checkpoint recovery defect. FLNA cells were transfected with an empty FLAG vector or a FLAG FLNA Bf constructs. Cells were mock treated (U) or treated with 8 Gy IR and cells were collected at different time points. Expression of FLAG FLNA Bf was unable to reverse the recove ry defect.
100 Figure 36. Expression of BRCA1 interacting fragment of FLNA phenocopies loss of FLNA. FLNA + cells were transfected with empty FLAG vector or a FLAG FLNA Bf constructs. Cells were mock treated (U) or treated with 8 Gy IR and cells were collected at different time points. Cells expressing of FLAG FLNA Bf di s played a phenotype similar to FLNA cells.
101 Figure 37. HCT166 cells stably expressing GFP FLNA Bf display a phenotype similar to FLNA cells. HCT116 cells were stably transfected with empty GFP vector or a GFP FLNA Bf constructs. Cells were mock treated (U) or treated with 8 Gy IR and cells were collected at different time points. Cells expressing of GFP FLNA Bf displayed a phenotype similar to FLNA cells.
102 Expression of BRCA1 Interacting Fragment of FLNA Phenocopies L oss of FLNA Next we asked whether expression of a GST tagged BRCA1 F LNA interacting f ragment (BRCA1 Ff) could also lead to a dominant n egative phenoty pe (Figure 38 ). In order to verify the specificity of the interaction we transfected a mutated BRCA1 Ff carrying the Y179C mutation and determined whether it lead to a dominant negative phen o type. Introduction of the Y179C mutation (Figure 1 9 ) significantly reduced the BRCA1 FLNA interaction. The wild type BRCA1 FLNA Ff led to increased and sustained pho s phorylation of CDC2 Y15 and H2AX (Figure 38 ) whi le the BRCA1 Ff Y179C (Figure 38 ) displayed a dominant negative effect that is intermediate between vector control (Figure 36 ) and the wild typ e construct (BRCA1 Ff; Figure 38 ). This intermediate effect could be due to the residual binding of BRCA1 Y179C mutant to FLNA. Alternatively, this could also be due to the inability of the mutant to disru pt the binding of FLNA to other regions of endogenous BRCA1 that participate in the interaction (Figure 16 ).
103 Figure 38. Expression of FLNA inte raction fragment of BRCA1 Y179C mutant does not phenocopy loss of FLNA. FLNA + cells were transfected with a GST BRCA1 Ff or a GST BRCA1 Ff Y179C. Cells were mock treated (U) or treated with 8 Gy IR and cells were collected at different time points. Only ce lls expressing of GST BRCA1 Ff but not GST BRCA1 Ff Y179C displayed a phenotype similar to FLNA cells, confirming that the effect is specific.
104 Analysis of the E ffec t of FLNA on DNA PKcs and Ku86 I nteraction FLNA is Required for Efficient Interactions B etween DN A PKcs and Ku86 Our previous experiments demonstrated that FLNA cells display defective DNA repair, showed signs of compromised HR, and accumulated large tracts of ssDNA. B e cause mammalian cells also repair DSBs using non homologous end joining (NHEJ) we hypothesized that lack of FLNA also had an impact on the NHEJ pathway. First, we tested whether FLAG FLNA aa 2477 2647 interacted with NHEJ factors. FLAG FLNA aa 2477 2647 immunoprecipitated DNA PKcs in 293FT cells ind e pendent of DNA damage (Figure 39 ). To determine whether FLNA was required for the stability of the Ku86/DNA PKcs complex we performed immunoprecipitation exper i ments in FLNA + and FLNA cell lines in the presence or a bsence of i rradiation (Figure 40 ). Interestingly, in FLNA cells Ku86 and D NA PKcs complex formation was co m promised in IR treate d and untreated cells (Figure 40 ). Next we tested whether FLNA was required for Ku86 loading onto chromatin a f ter DNA damage. Ku86 was efficiently recruited to chromatin upon DNA damage in the presenc e and abs ence of FLNA (Figure 41 ) while we detected DNA PKcs in chromatin only in the presence of FLNA (Figure 23 ). Interestingly, loading of Ku86 onto chromatin persisted longer and with consistently higher levels in FLNA than in FLNA + cells (Figure 41 ).
105 Finally, we tested whether BRCA1 was required to stabilize the interaction b e tween DNA PKcs and Ku86. As a model for studying BRCA1 functions in mammalian cells often is used HCC 1937 cell line. HCC 1937 cell line was established from 24 year old breast cancer patient, who is a carrier of BRCA1 5382insC mutation  and does not express p53 due to acquired p53 mutation  We examined BRCA1 deficient HCC1937 cell line  and a HCC1937 derivative reconstituted with full length BRCA1 (gift from J. Chen). Complex formation between Ku86 and DNA PKcs was not dependent on BRCA1 under IR treated or untreated conditions (Figure 42 ).
106 Figure 39. FLNA interacts in vivo with DNA PKcs. Upper pannel, 293FT cells were transfected with FLAG FLNA aa 2477 2647 (F) or empty FLAG vector (V) and mock treated or irradiated with 20 Gy. Cells were collected after 1h and immunopr e cipitated using FLAG antibody. FLAG FLNA aa 2477 2647 co immunoprecipitates DNA PKcs in the presence and absence of IR. Lower pa nel, co n trol for expression and the efficiency of the immunoprecipitation.
107 Figure 40. FLNA mediates DNA PKcs interaction to Ku86. The interaction b e tween DNA PKcs and Ku86 is compromised in cells lacking FLNA. Left panel shows that levels of DNA PKcs and Ku86 are similar in both cell lines and in the presence and absence of irradiation. Right panel shows that Ku86 and DNA PKcs interact in FLNA + cells in the absence of damage and complex formation is signif i cantly increased in the presence of irradiation. C omplex formation in the presence and absence of IR is severely compromised in FLNA cells.
108 Figure 41. Loading of Ku86 onto chromatin after DNA damage is increased in FLNA cells. FLNA + and FLNA cells were mock treated (U) or treated with 8 Gy IR and ce lls were collected at different time points. FLNA cells show increased amounts of Ku86 after IR. Histone H2AX levels are used as a loading control.
109 Figure 42. BRCA1 is not required for the stabilization of the interaction b e tween DNA PKcs and Ku86. HCC 1937 (BRCA1 ) and HCC1937 BRCA1wt (BRCA1 + ) were mock treated or treated with 20 Gy IR and cells were lyzed after 1h. DNA PKcs co immunoprecipitated with Ku86 independent of BRCA1 status.
110 Lack of FLNA Leads to an Increase in End Joining A ctivity Given the inability of DNA PKcs to interact with Ku86 and thus to be efficiently loaded on chromatin we decided to assess in vivo end joining activity. In order to mea s ure end joining we used the pGL2 plasmid that has a luciferase reporter gene driven by a constitutive promoter. First, we transfected FLNA and FLNA + cells with undigested pGL2 plasmid or d igested with either Hind III, which cuts the plasmid between the CMV promoter and the reporter gene, or Eco R1, which cuts the plasmid within the luciferase gene. Luciferase activity obtained by transfecting with the digested plasmids was mea s ured as a perce ntage of the activity obtained in cells transfected with the undigested plasmid. Luciferase activity obtained from the Hind III digested plasmid reflects total end joining activity, while that obtained from Eco R1 digested plasmid reflects precise end joinin g activity. Intriguingly, FLNA displayed up to 4 fold higher total and precise end joining activity compared to FLNA + cells (Figure 43 ). In summary, lack of FLNA leads to an increase in end joining activity on an episomal template.
111 Figure 43. Lack of Filamin A leads to an increase in end joining act ivity. FLNA and FLNA + cells were co transfected with a digested or undigested luciferase repor t er plasmid and an internal Renilla luciferase control. The reporter plasmid was either cut between the promoter and the reporter gene or inside the coding region for luc i ferase and recovered luciferase activity is a measure of total (left) or precise (right) end joining, respectively. On th e X axis the relative liciferase units are depicted.
112 DI SCUSSION In this work we shed light on the mechanism by which Filamin A (FLNA) is r e quired for efficient DNA repair. Our data indicates that lack of FLNA impacts on both homologous recombination and non homologous end joining FLNA is an actin binding pro tein and its inactivation leads to an array of disorders such as otopalatodigital spectrum disorder, Melnick Needles syndrome, and periventricular heterotopia  A l though of unclear significance, at least two families carrying germline mutations in BRCA1 have been shown to manifest ventricular heterotopia [237, 238] FLNA interacts with a variety of proteins, including BRCA2  and deficiency in FLNA leads to sensitivity to DNA damage and a defect in the recovery from G2 arrest  Thus, we i n vestigated further its role in the DNA damage response. FLNA bi nds BRCA1 using its extreme C terminus which contains its dimeriz a tion domain. BRCA1 interaction with FLNA is mediated by a 30 amino acid region in the N terminus of BRCA1 which contains a conserved domain called Motif 2  Intr o duction of the Y179C mutation in Motif 2 significantly decreases the interaction. Analy s es by the Align GV GD method or by a yeast based recombination assay suggest that Y179C may act as a deleterio us mutation [87, 239] On the other hand, this variant has been found co occurring in trans with a known deleterious mutation,
113 which indicates that it is unlikely to have severe effects  Thus, the Y179C may constitute a hypomorphic mutation with moderate effects on breast cancer predisposition. Of note, Motif 2 is close to the region that has been implicated in binding of BRCA1 to Ku86  In order to dissect the molecular role of FLNA in the DDR we took advantage of a well characterized genetically defined system. A melanoma cell line lacking FLNA was isolated and subsequently reconstituted with FLNA yielding a pair of cell lines in which the only difference is the presence or absence of FLNA  When we irradiated FLNA an d FLNA + cells, we noticed that FLNA took much lo nger to resolve DSBs (Fi g ures 21, 22 ). To elucidate the mechanism underlying the repair defect we systematically investigated the proficiency of damage signaling in FLNA cells. Initially we investigated t he recruitment and activation kinetics of the upstream kinases, as well as their downstream substrates after DNA damage. We found that FLNA deficiency led to the hyperactivation of ATM as judged by phosphorylation of ATM S1981 and CHK2 T68, surrogate marke rs of ATM activation [240 242] Similarly, lack of FLNA also led to a hyperactivation of ATR, as measured by CHK1 S317 phosphoryl a tion, a marker for ATR activation  Moreover, we also found sustained levels of phosphorylation of NBS1 S343 to be higher in FLNA cells. Although the role of NBS1 phosphorylation in the DNA damage signaling is poorly understood, it is generally thought to reflect ATM and ATR activation [199, 243] We also determined that major mediator protei ns BRCA1, MDC1, and 53BP1 formed IR induced foci irrespective of FLNA status. However, BRCA1 foci formation was significantly impaired
114 in FLNA deficient cells. In addition, Rad51 foci formation displayed a delayed kinetics in cells lacking FLNA. These data indicate that FLNA deficient cells have impaired homologous recombination. Indeed, Yue et al. showed that FLNA deficient cells have a reduced abil i ty to repair I SceI induced DSBs  During the course of our ex periments we noticed a consistent increase in the number of FLNA cells displaying IR induced RPA foci. These foci progressively increased in size at later time points after IR. RPA is a ssDNA binding protein and partic i pates in DNA metabolism processes wh ere there is generation of ssDNA such as replic a tion, repair, and recombination [244, 245] Phosphorylation leads to inability of RPA to associate with the replication centers and leads to the association with DNA d amage induced foci instead  In addition, we also verified the retention of phosphorylated RPA in the chromatin bound fraction of FLNA cells after treatment with CPT or HU. Consistently, we found more hyper phosphorylated forms of RPA associated with chr o matin in FLNA (Figures 27 29 ) In addition, the hyper phosphorylation of RPA persisted longer in FLNA (Figur es 27 29 ) Interestingly, lack of NHEJ proteins DNA PKc s and Ku86, which together with Ku70 form the active DNA PK complex, leads to accumul a tion of ssDNA in S phase  Thus, we further investigated how the lack of FLNA i m pacted on DNA PK complex formation. Remarkably, Ku86 failed to interact with DNA PKcs in the absence of FLNA. The reduc ed stability of the interaction is not due to Ku86 failure to load onto chromatin, as FLNA cells displayed sustained higher levels of chromatin bound Ku86 than FLNA + cells after damage. Ku86 is one of the first molecules to bind DNA ends after DSBs
115  and recruits DNA PKcs via i ts C terminus  Taken together these results establish that lack of FLNA results in an unstable association of Ku86 and DNA PKcs impairing the function of the complex. This impaired DNA PK activity leads to a c ont i nuous build up of ssDNA and Ku86 on chromatin. Over 16 phosphorylation sites have been identified in DNA PKcs although their role is still poorly understood. Nevertheless, DNA PKcs phosphorylation status is thought to influence its activity  DNA PKcs interacts with Ku86 and free ends of DNA in an unphosp horylated form  and autophosphorylation is required for NHEJ progression  Thus, we investigated the status of the two major phosphorylation clusters in DNA PKcs, namely the 2056 and 2609 clusters. Cluster 2609 was consistently phos phorylated upon treatment with IR, CPT and HU irrespective of the FLNA status. However, phosphorylation of the 2056 cluster was significantly reduced in FLNA cells in CPT but not in IR or HU treatment indicating that there are damage specific effects of D NA PKcs phosphorylation. Nevertheless, the fact that DNA PKcs is phosphorylated upon damage in the absence of FLNA suggests that DNA PKcs is interacting with the Ku86/DNA complex albeit transiently. Alternatively, it is possible that phosphorylation of DNA PKcs is not mediated by autophosphorylation at the synaptic complex but rather via hyperactive ATM and ATR in FLNA deficient cells. We showed that FLNA and BRCA1 interact and that FLNA deficiency leads to a marked decrease in BRCA1 foci formation after damage. To investigate further the role of BRCA1 we tested whether expression of the BRCA1 FLNA interacting fragment in FLNA proficient cells could also act in a dominant negative fashion leading to a
116 phen o type similar to FLNA deficient cells. Strikingly, expression of the BRCA1 Ff lead to a defect in DNA repair as judged by CDC2 pY15 and H2AX markers. This effect is sp e cific because expression of BRCA1 Ff containing a mutation that disrupts FLNA/BRCA1 interaction does not lead to the same phenotype. Take n together, these data establish that BRCA1 participates in the FLNA dependent regulation of the DNA damage response. Our data shows that absence of FLNA leads to defective DSB repair. The defect is a combined result of compromised HR and NHEJ processes. At this stage we cannot distinguish whether FLNA deficiency leads to a defective step that is common to both pathways or, alternatively, it impacts different steps in these pathways. In fact, the inte r play between these two arms of the DNA repair process is not fully understood  in particular after IR, which generates an array of different DNA modifications. The o b served phenotype is consistent with a model in which Ku86 recognizes and binds free ends of DNA, but in the absence of FLNA, fails to make a stable complex with DNA PKcs. We propose that unstable Ku86/DNA PKcs interaction results in impaired end processing, accumulation of ssDNA, and hyperactivation of DNA damage signaling. Repair via NHEJ can be arb itrarily divided into early (damage recognition and processing of non ligatable ends) and late (ligation) stages  DNA PK has multiple functions and is essential for early and late stages of classic al NHEJ in mammalian cells  To determine whether the lack of FLNA also impacted on the late stages of N HEJ we transfected cells with cleaved plasmids, which are thought not to require extensive processing, to monitor precise and total end joining activity. Cells lacking FLNA
117 di s played a significantly increased end joining activity, indicating that FLNA is n ot required for the late stages of end joining. The increase in end joining activity could be due to the fact that deficient DNA PK activity may lead to an unrepressed DNA ligase IV independent alternative end joining pathway [248 250] Alternatively, the failure of FLNA deficient cells to promote stable Ku86/DNA PKcs complex on chromatin may increase the availability of free DNA PKcs to act on the plasmid template. Further r e search will be needed to discriminate bet ween these possibilities. In addition, in FLNA deficient cells BRCA1 displays impaired foci formation suggesting that FLNA also plays a role in stabilizing BRCA1 at the DSBs. BRCA1 col o calizes with Rad50/Mre11/NBS1 complex at IR induced foci [120, 212] and inhibits Mre11 exonuclease activity  .Thus, the diminished amounts of BRCA1 at IR foci may lead to an unregulated Mre11 exonuclease activity with formation of the observed extended tracts of RPA coated ssDNA in FLNA deficient cells (Fig ures 31, 32, and 33 ) BRCA1 has also been implicated in the regulation of Rad51 [251, 252] although the m e chanism by which it happens is obscure  The kinetics of Rad51 foci formation in FLNA deficient cells suggests that there is no problem in the initial re cruitment to foci (see Figure 32 bottom panel, 3h time point). The extended plateau observed in Rad51 foci (from 3 to 12h aft er IR) may indicate an accumulation of DSBs that do not fulfill the end processing requirements for efficient Rad51 loading. Although further research will be needed to test this proposed model, it provides a tractable system to dissect the inte r play betwe en different processes involved in DNA repair.
118 It is possible that FLNA provides a framework for the assembly of factors in the synaptic complex. While unrepaired DNA in yeast (which lacks recognizable DNA PKcs and FLNA orthologs) migrates to so called DNA repair centers  the picture is di f ferent in mammalian cells where broken chromosome ends are essentially immobile [255, 256] It will be interesting to determine whet her lack of FLNA affects the mobility of broken ends.
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A BOUT THE AUTHOR Aneliya Velkova received a Bachelor of Science degree in Molecular Biology from the Sofia University (Bulgaria) in 2003. Afterwards, she worked as a research technician until 2005. She then pursued her graduate degree in the Cancer Biology Ph.D program at the University of South Florida and conducted research in the laboratory of Dr. Alvaro N.A. Monteiro at the H.Lee Moffitt Cancer Center and Research Institute. Three y ears of her graduate research were funded through a pre doctoral fel lowship from the Department of Defense Breast Cancer Research Program.